Single-sided light-actuated microfluidic device with integrated mesh ground

ABSTRACT

Single-sided optoelectrowetting (SSOEW)-configured substrates are provided, as well as microfluidic devices that include such substrates. The substrates can include a planar electrode, a photoconductive (or photosensitive) layer, a dielectric layer (single-layer or composite), a mesh electrode, and a hydrophobic coating. Fluid droplets can be moved across the hydrophobic coating of such substrates in a light-actuated manner, upon the application of a suitable AC voltage potential across the substrate and the focusing of light into the photoconductive layer of the substrate in a location proximal to the droplets. Walls can be disposed upon the substrates to form the microfluidic devices. Together the walls and substrate can form a microfluidic circuit, through which droplets can be moved.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application claims priority to, and the benefit of, U.S.provisional patent application Ser. No. 62/088,532 filed on Dec. 5,2014, incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable

TECHNICAL FIELD

The present invention relates generally to microfluidic devices havingan opto-eletrowetting (OEW) configuration. In particular, the presentdisclosure relates to single-sided opto-electrowetting (SSOEW) deviceshaving an integrated mesh ground.

BACKGROUND

Micro-objects, such as biological cells, can be processed inmicrofluidic devices. For example, droplets containing micro-objects orreagents can be moved around and merged within a microfluidic device.Embodiments of the present invention are directed to improvements inmicrofluidic devices that facilitate carrying out complex chemical andbiological reactions. Droplets can be moved and merged on or within amicrofluidic device by changing an effective wetting property of anelectrowetting surface in the microfluidic device. Such movements canfacilitate, for example, workflows in which cells are processed toassess various cellular properties. Microfluidic devices having anelectrowetting configuration typically include a hard cover, which cancomplicate the introduction of droplets onto and their removal from theelectrowetting surface. Accordingly, it is desirable to havemicrofluidic devices that have a more accessible electrowetting surface.

SUMMARY OF THE INVENTION

Embodiments of the present invention provide novel, single-sidedopto-electrowetting (SSOEW) devices having an integrated mesh ground.Any such SSOEW devices can form the base of a microfluidic device of theinvention, and the microfluidic device can optionally include channelsand/or chambers through which fluid can flow. The walls of the channelsand/or chambers can be attached at their base to the top surface of theSSOEW device, and the channels and/or chambers can be enclosed or openat their top.

Additional embodiments of the invention provide methods of moving one ormore droplets over the top surface of a SSOEW device of the invention.The one or more droplets (e.g., aqueous droplets) can have a volume thatranges from picoliters to microliters. Movement of a droplet can becontrolled by projecting structured electromagnetic radiation (e.g.,light) on a photoconductive layer of the SSOEW device, at a locationbeneath a leading edge of the droplet. The structured electromagneticradiation modulates the electro-wetting characteristics of the topsurface of the SSOEW device and can thereby generate a gradient in thesurface tension of the droplet. By changing the position of theprojected structured electromagnetic radiation, the droplet can be madeto follow the electromagnetic radiation (e.g., in a circular pattern, arectangular pattern, an irregular pattern, or any combination thereof).

Accordingly, in one aspect, the invention provides a substrate having aSSOEW configuration. The SSOEW-configured substrate can include a planarelectrode, a photoconductive layer, a dielectric layer, a mesh groundelectrode, and a hydrophobic coating. The photoconductive layer can beinterposed between the planar electrode and the dielectric layer, with abottom surface of the photoconductive layer adjoining a top surface ofthe planar electrode and a top surface of the photoconductive layeradjoining a bottom surface of the dielectric layer. The mesh groundelectrode can adjoin a top surface of the dielectric layer or can beembedded within the dielectric layer; and the hydrophobic coating cancoat the top surface of the dielectric layer and, depending upon itslocation, the top surface of the mesh ground electrode. The planarelectrode and the mesh ground electrode can be configured to beconnected to an AC voltage source (i.e., to opposing terminals of an ACvoltage source) such that, when so connected, the SSOEW-configuredsubstrate is capable of generating an optically actuated electrowetting(EW) force to aqueous droplets resting on or otherwise in contact withthe hydrophobic coating of the substrate. In certain embodiments, theSSOEW-configured substrate is part of a microfluidic device. In certainembodiments, the microfluidic device is a nanofluidic device.

In certain embodiments, the planar electrode includes a metal conductor.For example, the planar electrode can include an indium-tin-oxide (ITO)layer (e.g., ITO-coated glass). In certain embodiments, the planarelectrode comprises a non-metal conductor, such as conductive silicon(e.g., highly n- or p-doped silicon). Regardless, the metal or non-metalconductor should be thick enough to effectively conduct electricalcurrent. Typically, a layer of conductive silicon will be at least 500microns (e.g., about 500 to 1000 microns, about 550 to 800 microns, orabout 600 to 700 microns) thick. In certain embodiments, the planarelectrode includes both metal conductor and non-metal conductorelements. For example, the planar electrode can include a layer ofconductive silicon with a layer of conductive metal (e.g., gold, silver,ITO) on the underside of the conductive silicon layer.

In certain embodiments, the photoconductive layer comprises hydrogenatedamorphous silicon (a-Si:H). The photoconductive layer can have athickness of at least 100 nm (e.g., at least 250 nm, 500 nm, 750 nm,1000 nm, or more). In certain embodiments, the photoconductive layer canhave a thickness of about 500 nm to 1500 nm (e.g., about 600 nm to about1400 nm, about 700 nm to about 1300 nm, about 800 nm, to about 1200 nm,about 900 nm to about 1100 nm, or about 1000 nm).

In certain embodiments, the dielectric layer comprises a metal oxide,such as aluminum oxide or hafnium oxide. In certain embodiments, thedielectric layer has a thickness of at least 50 nm (e.g., at least about75 nm, 100 nm, 125 nm, 150 nm, or more). For example, the dielectriclayer can have a thickness of about 50 to about 250 nm (e.g., about 75nm to about 225 nm, or about 100 nm to about 200 nm, or about 125 nm toabout 175 nm, or about 140 nm to about 160 nm). In certain embodiments,the dielectric layer has been formed by atomic layer deposition. Incertain embodiments, the dielectric layer has an impedance of about 10to 50 kOhms.

In certain embodiments, the dielectric layer is a composite dielectriclayer. For example, the dielectric layer can have at least a firstdielectric layer and a second dielectric layer. The bottom surface ofthe first dielectric layer can be adjoining the photoconductive layer.In certain embodiments, the mesh ground electrode is interposed betweenthe first dielectric layer and the second dielectric layer. Thecomposite dielectric layer can have a thickness of at least about 50 nm(e.g., at least about 75 nm, 100 nm, 125 nm, 150 nm, or more). Forexample, the dielectric layer can have a thickness of about 50 to about250 nm (e.g., about 75 nm to about 225 nm, about 100 nm to about 200 nm,about 125 nm to about 175 nm, or about 140 nm to 160 nm). In certainembodiments, the composite dielectric layer has an impedance of about 10to 50 kOhms.

In certain embodiments, the each of the first and second dielectriclayers of a composite dielectric layer comprise a metal oxide, such asaluminum oxide or hafnium oxide. One or both of the first and seconddielectric layers can be formed by atomic layer deposition. In suchembodiments, the first dielectric layer can have a thickness of at leastabout 100 nm (e.g., at least about 125 nm, about 150 nm, or more). Forexample, the first dielectric layer can have a thickness of about 125 nmto about 175 nm (e.g., about 140 nm to about 160 nm). In suchembodiments, the second dielectric layer can have a thickness of about50 nm or less (e.g., about 40 nm, about 30 nm, about 25 nm, about 20 nm,about 15 nm, about 10 nm, about 9 nm, about 8 nm, about 7 nm, about 6nm, about 5 nm, about 4 nm, about 3 nm, or less). In certainembodiments, the first dielectric layer has a thickness of at least 125nm (e.g., at least 150 nm) and the second dielectric layer has athickness of 10 nm or less than 10 nm, (e.g., 5 nm or less).

In certain embodiments, the first dielectric layer of a compositedielectric layer has a lattice shape of substantially uniform thickness,with a top surface of the first dielectric layer adjoining a bottomsurface of the mesh ground electrode and the mesh ground electrodeinterposed between the first dielectric layer and the second dielectriclayer. The top surface of the first dielectric layer can besubstantially contiguous with the bottom surface of the mesh groundelectrode. In such embodiments, the first dielectric layer can comprisea metal oxide, such as aluminum oxide or hafnium oxide, and can have athickness of at least about 50 nm (e.g., at least about 75 nm, about 100nm, about 125 nm, about 150 nm, or more). For example, the firstdielectric layer can have a thickness of about 50 nm to about 200 nm(e.g., about 75 nm to about 180 nm, about 100 nm to about 160 nm, orabout 125 nm to about 150 nm). In such embodiments, the seconddielectric layer can comprise a non-metal oxide, such as silicon oxide,and can have a variable thickness. For example, the second dielectriclayer can have first regions, contacting the top surface of thephotoconductive layer, having a thickness substantially the same as thethickness of the composite dielectric layer, and second regions,directly over the wires of the mesh ground electrode, having a thicknessof about 50 nm or less (e.g., about 40 nm or less, about 30 nm or less,about 25 nm or less, about 20 nm or less, about 15 nm or less, about 10nm or less, about 9 nm or less, about 8 nm or less, about 7 nm or less,about 6 nm or less, about 5 nm or less, about 4 nm or less, or about 3nm or less). In certain embodiments, the first dielectric layer has athickness of at least 125 nm (e.g., at least 150 nm) and the seconddielectric layer has a thickness, directly over the wired of the meshground electrode of 10 nm or less (e.g., 5 nm or less).

In certain embodiments, the first dielectric layer of a compositedielectric layer is made from a first material having a dielectricconstant ∈₁ and the second dielectric layer is made from a secondmaterial having a dielectric constant ∈₂, where ∈₁ is different than ∈₂.For example, ∈₁ can be less than ∈₂. For example, the first material canbe a metal oxide, such as aluminum oxide or hafnium oxide, and thesecond material can be a non-metal oxide, such as silicon oxide.

In certain embodiments, the composite dielectric layer comprises a firstdielectric layer, a second dielectric layer, and a third dielectriclayer. The second dielectric layer can be interposed between the firstand third dielectric layers. In certain embodiments, the first and thirddielectric layers each comprise a metal oxide, such as aluminum oxide.In such embodiments, one or both of the first and third dielectriclayers may have been formed by atomic layer deposition. The firstdielectric layer can have a thickness of at least about 10 nm (e.g.,about 10 nm to about 20 nm). In addition, the third dielectric layer canhave a thickness of at least about 10 nm (e.g., about 10 nm to about 20nm). In related embodiments, the second dielectric layer can comprise anon-metal oxide or a nitride. The non-metal oxide can be, for example,silicon oxide. The second dielectric layer can be formed by plasmaenhanced chemical vapor deposition and can have a thickness of at leastabout 75 nm (e.g., at least about 100 nm, at least about 125 nm, atleast about 150 nm, or more). For example, the second dielectric layercan have a thickness of about 100 nm to about 200 nm (e.g., about 125 nmto about 175 nm).

In certain embodiments, the first dielectric layer of a three-layercomposite dielectric layer has a top surface adjoining a bottom surfaceof the mesh ground electrode, and the third dielectric layer has abottom surface adjoining a top surface of the mesh ground electrode.Thus, for example, the mesh ground electrode can be entirely encasedwithin the composite dielectric layer, with the second dielectric layerfilling the spaces formed between the lateral edges of the wires of themesh ground electrode.

In certain embodiments, the first dielectric layer of a three-layercomposite dielectric layer is made from a first material that has adielectric constant the second dielectric layer is made from a secondmaterial that has a dielectric constant ∈₂, the third dielectric layeris made from a third material that has a dielectric constant ∈₃, and ∈₁is different than ∈₃. For example, ∈₁ can be less than ∈₃. In certainrelated embodiments, ∈₂ can be less than ∈₃ (e.g., ∈₂ can have a valueequal to or greater than ∈₁ but less than ∈₃). In certain embodiments,the three-layer composite dielectric layer has an impedance of about 10to 50 kOhms.

In certain embodiments, the mesh ground electrode comprises a pluralityof wires arranged in a lattice shape. The mesh ground electrode canfurther comprise plates located on top of vertices formed by wires ofthe mesh ground electrode. The plates, for example, can havesubstantially the same composition as the wires of the mesh groundelectrode. In certain embodiments, the wires of the mesh groundelectrode have a substantially square shape or a substantiallyrectangular shape (viewed in cross-section). Thus, the wires can have anaverage width and an average height. The average height of the wires canbe at least about 50 nm (e.g., at least about 60 nm, about 70 nm, about80 nm, about 90 nm, about 100 nm, or more). The average width of thewires can be at least about 100 nm (e.g., at least about 200 nm, about300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about800 nm, about 900 nm, about 1000 nm, or more). In other embodiments, thewires of the mesh ground electrode can have a T-shape (viewed in crosssection).

In certain embodiments, the mesh ground electrode comprises a conductivematerial, such as a metal (e.g., gold, aluminum, chromium, titanium, orcombinations thereof). In certain embodiments, the mesh ground electrodecomprises a layer of gold and an underlying layer of chromium ortitanium. In certain embodiments, the conductive material is asemiconductor material that is highly electrically conductive (e.g.,highly-doped silicon). In certain embodiments, the conductive materialof the mesh ground electrode has an oxidized outer surface. For example,the mesh ground electrode can comprise aluminum that has an oxidizedouter surface.

In certain embodiments, the mesh ground electrode has a linear fillfactor β that is less than or equal to about 10% (e.g., about 9%, about8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about1%, about 0.5%, or less). In certain embodiments, the wires of the meshground electrode have a pitch of less than 1 mm (e.g., about 200 micronsto about 500 microns). In certain embodiments, the wires of the meshground electrode have a pitch of about 1.5 mm to 2 mm, about 1.0 mm to1.5 mm, about 0.5 mm to 1.0 mm, about 400 to 800 microns, about 300 to600 microns, about 200 to 400 microns, about 100 to 200 microns, about50 to 100 microns, or about 10 to 50 microns.

In certain embodiments, the hydrophobic coating has a bottom surfacethat adjoins some or all of the top surface of the dielectric layer. Incertain related embodiments, the bottom surface of the hydrophobiccoating adjoins the top surface (and, optionally, the lateral surfacesof the wires) of the mesh ground electrode. Thus, the bottom surface ofthe hydrophobic coating can adjoin both the top surface of thedielectric layer and surfaces of the mesh ground electrode that do notadjoin the dielectric layer.

In certain embodiments, the hydrophobic coating can comprise anorganofluorine polymer. The organofluorine polymer can have, forexample, at least one perfluorinated segment. The organofluorine polymercan comprise, for example, polytetrafluoro-ethylene (PTFE).Alternatively, the organofluorine polymer can comprisepoly(2,3-difluoromethylenyl-perfluorotetrahydrofuran). In suchembodiments, the hydrophobic coating can have a thickness of at leastabout 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about35 nm, or more.

In certain embodiments, the hydrophobic layer comprises a densely packedmonolayer of amphiphilic molecules covalently bonded to molecules of thedielectric layer. The amphiphilic molecules of the hydrophobic layer caneach comprise a siloxane group, a phosphonic acid group, or a thiolgroup, and the respective siloxane groups, phosphonic acid groups, andthiol groups can be covalently bonded to the molecules of the dielectriclayer. In such embodiments, the hydrophobic layer can have a thicknessof less than 5 nm (e.g., a thickness of about 1.5 to 3.0 nm).

In certain embodiments, the amphiphilic molecules of the hydrophobiclayer comprise long-chain hydrocarbons. Thus, for example, theamphiphilic molecules can be alkyl-terminated siloxane, alkyl-terminatedphosphonic acid, or alkyl-terminated thiol molecules. The long chainhydrocarbons can be unbranched, and can comprise a chain of at least 10carbons (e.g., at least 16, 18, 20, 22, or more carbons). Thealkyl-terminated siloxane can comprise trimethoxysilane,triethoxysilane, or triclorosilane. For example, the alkyl-terminatedsiloxane can be selected from the group consisting ofoctadecyltrimethoxysilane, octadecyltriethoxysilane, oroctadecyltriclorosilane. In certain embodiments, the amphiphilicmolecules of the hydrophobic layer can comprise fluorinated carbonchains. The fluorinated carbon chains have the chemical formulaCF₃—(CF₂)_(m)—(CH₂)_(n)—, wherein m is at least 2, n is at least 2, andm+n is at least 9. For example, m can be 7 (or greater) and n can be 2(or greater). In certain embodiments, the hydrophobic layer is patternedsuch that select regions are relatively hydrophilic compared to theremainder of the hydrophobic layer.

In another aspect, the invention provides a microfluidic device having abase that includes a substrate having a SSOEW configuration, and wallsdisposed on the base. The base and the walls can together define amicrofluidic circuit. The microfluidic device can optionally include acover, which can be disposed on the walls. The SSOEW-configuredsubstrate of the base can be any of the SSOEW-configured substratesdescribed herein.

In certain embodiments, the walls comprise a structural polymer. Thepolymer can be a silicon-based polymer, such as polydimethylsiloxane(PDMS) or photo-patternable silicone (PPS). Alternatively, the walls cancomprise an epoxy-based adhesive, such as SU-8. For microfluidic devicesthat include a cover, the cover can comprise the same structural polymercontained in the walls. In addition, the cover can be integral with thewalls. Alternatively, the cover can comprise a material different thanthe walls. Whether or not a cover is present, the walls can have aheight of at least 30 microns (i.e., the walls can rise above the baseby at least 30 microns). For example, the walls can have a height ofabout 40 to about 100 microns.

In certain embodiments, the microfluidic circuit can include one or more(e.g., 2, 3, 4, 5, 6, 7, or more) microchannels. In addition, themicrofluidic circuit can include a plurality of chambers (e.g.,sequestration pens). The chambers can open off of one (or more) of themicrochannels. In certain embodiments, the chamber can include a holdingregion configured to hold a liquid droplet, and at least one connectionregion that fluidically connects the holding region to a microfluidicchannel. A first connection region can be configured to allow movementof the liquid droplet between the microfluidic channel and the chamber.A second connection region, if present, can be configured to allow forfluid flow and pressure relief when a liquid droplet is moved betweenthe microfluidic channel and the holding region. In certain embodiments,the chamber can be connected to both a first microfluidic channel and asecond microfluidic channel.

In certain embodiments, the microfluidic channel(s) can have a width ofabout 50 to about 1000 microns, or about 100 to about 500 microns, withthe width measured in a direction normal to the direction of fluid flowthrough the channel. In certain embodiments, the chamber (orsequestration pen) can a cross-sectional area of about 100,000 to about2,500,000 square microns, or about 200,000 to about 2,000,000 squaremicrons. In certain embodiments, the connection region has a width ofabout 50 to about 500 microns, or about 100 to about 300 microns.

In certain embodiments, at least a portion of the base that helps todefine the microchannel(s) and/or the chamber(s) can have a SSOEWconfiguration. The SSOEW configuration can be connected to a biasingpotential and, while thus connected, change an effective wettingcharacteristic of any of a plurality of corresponding regions of thebase surface. The wetting characteristic of the base surface can bechanged sufficiently to move a liquid droplet across the substratesurface and between the microfluidic channel and the chamber.

In certain embodiments, the microfluidic device can include one or moreculture chambers suitable for culturing biological micro-objects. Theculture chamber(s) can be located within the microfluidic circuit, andeach one can be fluidically connected to one or more microfluidicchannels. For example, a culture chamber can be fluidically connected toa first microfluidic channel which is configured for moving droplets toand/or from the culture chamber, and a second microfluidic channel thatis configured to flow fresh culture medium past the culture chamber suchthat nutrients in the fresh culture medium and waste products in theculture chamber can be exchanged (e.g., by diffusion of nutrients intothe culture chamber and diffusion of waste products out into the culturemedium). The second channel can be separate from the first channel.

In another aspect, the invention provides methods for moving a dropletof fluid in a microfluidic device. The methods can include disposing adroplet of an aqueous solution on a top surface of a base of amicrofluidic device, applying an AC voltage potential between a planarelectrode and a mesh ground electrode of the microfluidic device,directing structured light at a position on the top surface of the baseof the microfluidic device, in a location proximal to the droplet ofaqueous solution, and moving the structured light relative to themicrofluidic device at a rate that induces the droplet of aqueoussolution to move across the top surface of the base. The microfluidicdevice can comprise a SSOEW-configured substrate, such as describedherein.

In certain embodiments, the droplet of aqueous solution has a volume ofabout 1 microliter or less, about 900 nL or less, about 800 nL or less,about 700 nL or less, about 600 nL or less, about 500 nL or less, about400 nL or less, about 300 nL or less, about 200 nL or less, about 100 nLor less, or about 50 nL or less. Alternatively, the droplet of aqueoussolution can have a volume of greater than 1 microliter (e.g., at leastabout 1.1 microliters, at least about 1.2 microliters, at least about1.3 microliters, at least about 1.4 microliters, at least about 1.5microliters, at least about 1.6 microliters, at least about 1.7microliters, at least about 1.8 microliters, at least about 1.9microliters, at least about 2.0 microliters, at least about 2.2microliters, at least about 2.4 microliters, at least about 2.6microliters, at least about 2.8 microliters, at least about 3.0microliters, or more). The droplet can have, for example, a conductivityof at least 0.1 mS/m (e.g., at least 1 mS/m). In certain embodiments,the AC voltage potential applied between the planar electrode and themesh ground electrode of the microfluidic device is about 10 ppV toabout 80 ppV (e.g., about 30 ppV to about 50 ppV). In certainembodiments, the AC voltage potential applied between the planarelectrode and the mesh ground electrode of the microfluidic device has afrequency of about 1 kHz to about 1 MHz (e.g., about 1 kHz to about 100kHz, about 2 kHz to about 80 kHz, about 3 kHz to about 60 kHz, about 4kHz to about 40 kHz, or about 5 kHz to about 20 kHz). In certainembodiments, the structured light is moved relative to the microfluidicdevice at a rate of 0.05 cm/sec or greater (e.g., 0.1 cm/sec or greater,0.2 cm/sec or greater, or 0.3 cm/sec or greater) and the droplet moveswith the light.

In another aspect, the invention provides methods of processing dropletsin a microfluidic device. The methods include filling a microfluidiccircuit of a microfluidic device with a first liquid, applying an ACvoltage potential between a planar electrode and a mesh ground electrodeof the microfluidic device, introducing a first droplet of liquid mediuminto the microfluidic circuit, wherein the first droplet is immisciblein the first liquid medium, and moving the first droplet to a desiredlocation within the microfluidic circuit by applying an electrowetting(EW) force to the first droplet. The microfluidic device can be any ofthe microfluidic devices described herein (e.g., a microfluidic devicehaving a base that comprises a SSOEW-configured substrate). In addition,the microfluidic device comprises a droplet generator. The dropletgenerator can be configured to selectively provide droplets of one ormore liquid media into the microfluidic circuit of the microfluidicdevice.

In certain embodiments, the applied AC voltage potential is about 10 ppVto about 80 ppV. For example, the applied AC voltage potential can beabout 30 ppV to about 50 ppV. In addition, the applied AC voltagepotential can have a frequency of about 1 to 100 kHz (e.g., about 1 kHzto about 100 kHz, about 2 kHz to about 80 kHz, about 3 kHz to about 60kHz, about 4 kHz to about 40 kHz, or about 5 kHz to about 20 kHz).

In certain embodiments, the first liquid medium is an oil. For example,the first liquid medium is a silicone oil, a fluorinated oil, or acombination thereof. The first droplet can comprise an aqueous solution.The aqueous solution can be, for example, a saline solution or a cellculture medium.

In certain embodiments, the first droplet of liquid medium can compriseat least one micro-object. For example, the first droplet can comprise abiological micro-object (e.g., a single cell) or a capture bead (e.g., 1to 20 capture beads). The capture bead can have an affinity for amaterial of interest, such as a biological cell secretion or a nucleicacid (e.g., DNA, genomic DNA, mitochondrial DNA, RNA, mRNA, miRNA, orany combination thereof).

In certain embodiments, the first droplet of liquid medium can comprisea reagent. The reagent can be a lysis buffer, a fluorescent label, aluminescent assay reagent, or the like. The lysis buffer can include anon-ionic detergent (e.g., at a concentration of less than 0.2%) or aproteolytic enzyme. The proteolytic enzyme can be inactivatable (e.g.,by heat or specific inhibitor).

In certain embodiments, the methods of processing droplets in amicrofluidic device further include: introducing a second droplet ofliquid medium into the microfluidic circuit, wherein the liquid of thesecond droplet is immiscible in the first liquid medium but misciblewith the liquid medium of the first droplet; moving the second dropletto a location within the microfluidic circuit adjacent to the firstdroplet by applying an EW force to the second droplet; and merging thesecond droplet with the first droplet to form a combined droplet. Thesecond droplet can be merged with the first droplet by applying an EWforce to the second and/or the first droplet.

In certain embodiments, the first droplet comprises a biologicalmicro-object and the second droplet comprises a reagent. The reagentcontained in the second droplet can be selected from the group consistof a lysis buffer, a fluorescent label, and a luminescent assay reagent.For example, the reagent contained in the second droplet can be a lysisbuffer, and the biological micro-object (e.g., cell) in the firstdroplet can be lysed upon merger of the first droplet and the seconddroplet.

In some embodiments, the methods of processing droplets in amicrofluidic device further include introducing third, fourth, etc.droplets into the microfluidic circuit and moving the third, fourth,etc. droplet to a desired location within the microfluidic circuit byapplying an EW force to the droplet. The third droplet can be moved to aposition proximal to the first combined droplet and then merged with thefirst combined droplet to form a second combined droplet; the fourthdroplet can be moved to a position proximal to the second combineddroplet and then merged with the second combined droplet to form a thirdcombined droplet; and so on. Each additional droplet can contain afluidic medium that is immiscible in the first liquid medium butmiscible with the liquid medium of the first droplet (and/or first,second, etc. combined droplet).

In some embodiments, the first droplet can contain a biological cell andthe second droplet can contain a reagent. The reagent can be a celllysis buffer that lyses the biological cell when the first and seconddroplets are merged. Alternatively, the reagent can be a fluorescentlabel (e.g., a fluorescently-labeled antibody or other affinity reagent)or a reagent used in a luminescence assay. The third droplet can containa reagent, such as one or more (e.g., two to twenty) capture beadshaving affinity for a material of interest. For example, the material ofinterest can be an antibody or a nucleic acid, such as DNA, genomic DNA,mitochondrial DNA, RNA, mRNA, miRNA, or any combination thereof. Suchcapture beads can optionally be exported from the apparatus forsubsequent analysis. The fourth droplet can, like the second and thirddroplets, contain a reagent, such as an enzymatic mixture suitable forperforming a reaction, such as a reverse transcriptase reaction or awhole genome amplification reaction.

In certain embodiments, applying an EW force to move and/or mergedroplets comprises changing an effective electrowetting characteristicof a region of the top surface of the base of the microfluidic deviceproximal to the droplet(s). Changing the effective electrowettingcharacteristic can include activating EW electrodes (e.g., regions inthe photoconductive layer) in the base of the microfluidic device at alocation proximal to the droplet(s). Activating the EW electrodes in thebase of the microfluidic device proximal to the droplet(s) can includedirecting a pattern of light onto the photoconductive layer of the baseat the location proximal to the droplet(s).

Further aspects of the technology described herein will be brought outin the drawings and the following portions of the specification, whereinthe detailed description is for the purpose of fully disclosingpreferred embodiments of the technology without placing limitationsthereon.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an example of a system and associated controlequipment for controlling a microfluidic device, which can be asingle-sided opto-electrowetting (SSOEW) device.

FIG. 2A illustrates a specific example of a support which can be part ofthe system and can be configured to hold and operatively couple with amicrofluidic device.

FIG. 2B illustrates an imaging device which can be part of the system.

FIGS. 3A and 3B illustrate a cut-away view (FIG. 3A) and across-sectional view (FIG. 3B) of a portion of a SSOEW device accordingto one embodiment of the invention.

FIG. 4A illustrates the SSOEW device of FIG. 3B overlaid with anelectrical circuit diagram showing resistive and capacitive elements ofthe SSOEW device when an aqueous droplet is positioned on the surface ofthe device and a voltage source is placed between a bottom electrode andan upper mesh electrode. FIG. 4B provides an isolated view of theelectrical circuit diagram of FIG. 4A.

FIGS. 5A and 5B illustrate a cut-away view (FIG. 5A) and across-sectional view (FIG. 5B) of a portion of a single-sided OEW deviceaccording to one embodiment of the invention.

FIGS. 6A-6C illustrate alternative embodiments for a meshelectrode-containing dielectric layer of the SSOEW device of FIGS. 5Aand 5B. FIG. 6A shows a cross-sectional view of the dielectric layerwhich has “T” shaped wires that make up the mesh electrode. FIG. 6Bshows a top-down view of a mesh electrode that has square caps locatedat each vertex formed by the crossing of two wires. FIG. 6C shows across-sectional view of the dielectric layer in which the wires of themesh electrode are supported by a dielectric material that is differentfrom the dielectric material that flanks and covers the mesh electrode.

FIG. 7 is a top-down view of a microfluidic apparatus having multiplemicrofluidic channels, chambers that open off of the microfluidicchannels, and a droplet generator, according to certain embodiments ofthe invention. In this embodiment, one microfluidic channel contains anaqueous medium (lighter color), while the microfluidic channel connectedto the droplet generator contains a hydrophobic medium (darker color).The chambers likewise contain either an aqueous medium or a hydrophobicmedium.

FIG. 8 is a top-down view of a microfluidic apparatus having multiplemicrofluidic channels, chambers that open off of the microfluidicchannels, and a droplet generator, according to other embodiments of theinvention.

FIG. 9 is a graph showing estimates of the minimum volume of a dropletthat can be moved with a SSOEW-configured substrate having a mesh groundelectrode with a given pitch. The graph also shows the maximum wirewidth that provides >90% EW force versus a given pitch.

FIG. 10 shows images of droplet movement on a SSOEW-configured substrateaccording to certain embodiments of the invention. The images depictboth rectangular movements (a(i)-(iii)) and circular movements(b(i)-(iii)).

DETAILED DESCRIPTION OF THE INVENTION

This specification describes exemplary embodiments and applications ofthe invention. The invention, however, is not limited to these exemplaryembodiments and applications or to the manner in which the exemplaryembodiments and applications operate or are described herein. Moreover,the figures may show simplified or partial views, and the dimensions ofelements in the figures may be exaggerated or otherwise not inproportion. In addition, as the terms “on,” “attached to,” “connectedto,” “coupled to,” or similar words are used herein, one element (e.g.,a material, a layer, a substrate, etc.) can be “on,” “attached to,”“connected to,” or “coupled to” another element regardless of whetherthe one element is directly on, attached to, connected to, or coupled tothe other element or there are one or more intervening elements betweenthe one element and the other element. In addition, where reference ismade to a list of elements (e.g., elements a, b, c), such reference isintended to include any one of the listed elements by itself, anycombination of less than all of the listed elements, and/or acombination of all of the listed elements.

Section divisions in the specification are for ease of review only anddo not limit any combination of elements discussed.

As used herein, “substantially” means sufficient to work for theintended purpose. The term “substantially” thus allows for minor,insignificant variations from an absolute or perfect state, dimension,measurement, result, or the like such as would be expected by a personof ordinary skill in the field but that do not appreciably affectoverall performance. When used with respect to numerical values orparameters or characteristics that can be expressed as numerical values,“substantially” means within ten percent.

As used herein, the term “ones” means more than one. As used herein, theterm “plurality” can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

As used herein, the term “disposed” encompasses within its meaning“located.”

As used herein, a “microfluidic device” or “microfluidic apparatus” is adevice that includes one or more discrete microfluidic circuitsconfigured to hold a fluid, each microfluidic circuit comprised offluidically interconnected circuit elements, including but not limitedto region(s), flow path(s), channel(s), chamber(s), and/or pen(s).Certain microfluidic devices (e.g., those that include a cover) willfurther include at least two ports configured to allow the fluid (and,optionally, micro-objects or droplets present in the fluid) to flow intoand/or out of the microfluidic device. Some microfluidic circuits of amicrofluidic device will include at least one microfluidic channeland/or at least one chamber. Some microfluidic circuits will hold avolume of fluid of less than about 1 mL, e.g., less than about 750, 500,250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2μL. In certain embodiments, the microfluidic circuit holds about 1-2,1-3, 1-4, 1-5, 2-5, 2-8, 2-10, 2-12, 2-15, 2-20, 5-20, 5-30, 5-40, 5-50,10-50, 10-75, 10-100, 20-100, 20-150, 20-200, 50-200, 50-250, or 50-300μL.

As used herein, a “nanofluidic device” or “nanofluidic apparatus” is atype of microfluidic device having a microfluidic circuit that containsat least one circuit element configured to hold a volume of fluid ofless than about 1 μL, e.g., less than about 750, 500, 250, 200, 150,100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nL or less.Typically, a nanofluidic device will comprise a plurality of circuitelements (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75,100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000,2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, ormore). In certain embodiments, one or more (e.g., all) of the at leastone circuit elements is configured to hold a volume of fluid of about100 pL to 1 nL, 100 pL to 2 nL, 100 pL to 5 nL, 250 pL to 2 nL, 250 pLto 5 nL, 250 pL to 10 nL, 500 pL to 5 nL, 500 pL to 10 nL, 500 pL to 15nL, 750 pL to 10 nL, 750 pL to 15 nL, 750 pL to 20 nL, 1 to 10 nL, 1 to15 nL, 1 to 20 nL, 1 to 25 nL, or 1 to 50 nL. In other embodiments, oneor more (e.g., all) of the at least one circuit elements is configuredto hold a volume of fluid of about 100 to 200 nL, 100 to 300 nL, 100 to400 nL, 100 to 500 nL, 200 to 300 nL, 200 to 400 nL, 200 to 500 nL, 200to 600 nL, 200 to 700 nL, 250 to 400 nL, 250 to 500 nL, 250 to 600 nL,or 250 to 750 nL.

A “microfluidic channel” or “flow channel” as used herein refers to flowregion of a microfluidic device having a length that is significantlylonger than the horizontal dimension (and vertical dimension, if themicrofluidic device includes a cover). For example, the flow channel canbe at least 5 times the length of either the horizontal (or vertical)dimension, e.g., at least 10 times the length, at least 25 times thelength, at least 100 times the length, at least 200 times the length, atleast 500 times the length, at least 1,000 times the length, at least5,000 times the length, or longer. In some embodiments, the length of aflow channel is in the range of from about 100,000 microns to about500,000 microns, including any range therebetween. In some embodiments,the horizontal dimension is in the range of from about 100 microns toabout 1000 microns (e.g., about 150 to about 500 microns) and, ifpresent, the vertical dimension is in the range of from about 25 micronsto about 200 microns, e.g., from about 40 to about 150 microns. It isnoted that a flow channel may have a variety of different spatialconfigurations in a microfluidic device, and thus is not restricted to aperfectly linear element. For example, a flow channel may be, or includeone or more sections having, the following configurations: curve, bend,spiral, incline, decline, fork (e.g., multiple different flow paths),and any combination thereof. In addition, a flow channel may havedifferent cross-sectional areas along its path, widening andconstricting to provide a desired fluid flow therein.

As used herein, the term “obstruction” refers generally to a bump orsimilar type of structure that is sufficiently large so as to partially(but not completely) impede movement of target micro-objects between twodifferent regions or circuit elements in a microfluidic device. The twodifferent regions/circuit elements can be, for example, a microfluidicsequestration pen and a microfluidic channel, or between a connectionregion and an isolation region of a microfluidic sequestration pen.

As used herein, the term “constriction” refers generally to a narrowingof a width of a circuit element (or an interface between two circuitelements) in a microfluidic device. The constriction can be located, forexample, at the interface between a microfluidic sequestration pen and amicrofluidic channel, or at the interface between an isolation regionand a connection region of a microfluidic sequestration pen.

As used herein, the term “transparent” refers to a material which allowsvisible light to pass through without substantially altering the lightas is passes through.

As used herein, the term “micro-object” refers generally to anymicroscopic object that may be manipulated in accordance with thepresent invention. Non-limiting examples of micro-objects include:inanimate micro-objects such as microparticles; microbeads (e.g.,polystyrene beads, Luminex™ beads, or the like); magnetic beads;microrods; microwires; quantum dots, and the like; biologicalmicro-objects such as cells (e.g., embryos, oocytes, sperm cells, cellsdissociated from a tissue, eukaryotic cells, protist cells, animalcells, mammalian cells, human cells, immunological cells, hybridomas,cultured cells, cells from a cell line, cancer cells, infected cells,transfected and/or transformed cells, reporter cells, prokaryotic cell,and the like); biological organelles; vesicles, or complexes; syntheticvesicles; liposomes (e.g., synthetic or derived from membranepreparations); lipid nanorafts (as described in Ritchie et al. (2009)“Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs,”Methods Enzymol., 464:211-231), and the like; or a combination ofinanimate micro-objects and biological micro-objects (e.g., microbeadsattached to cells, liposome-coated micro-beads, liposome-coated magneticbeads, or the like). Beads may further have other moieties/moleculescovalently or non-covalently attached, such as fluorescent labels,proteins, small molecule signaling moieties, antigens, orchemical/biological species capable of use in an assay.

As used herein, the term “maintaining (a) cell(s)” refers to providingan environment comprising both fluidic and gaseous components and,optionally a surface, that provides the conditions necessary to keep thecells viable and/or expanding.

A “component” of a fluidic medium is any chemical or biochemicalmolecule present in the medium, including solvent molecules, ions, smallmolecules, antibiotics, nucleotides and nucleosides, nucleic acids,amino acids, peptides, proteins, sugars, carbohydrates, lipids, fattyacids, cholesterol, metabolites, or the like.

As used herein in reference to a fluidic medium, “diffuse” and“diffusion” refer to thermodynamic movement of a component of thefluidic medium down a concentration gradient.

The phrase “flow of a medium” means bulk movement of a fluidic mediumprimarily due to any mechanism other than diffusion. For example, flowof a medium can involve movement of the fluidic medium from one point toanother point due to a pressure differential between the points. Suchflow can include a continuous, pulsed, periodic, random, intermittent,or reciprocating flow of the liquid, or any combination thereof. Whenone fluidic medium flows into another fluidic medium, turbulence andmixing of the media can result.

The phrase “substantially no flow” refers to a rate of flow of a fluidicmedium that, averaged over time, is less than the rate of diffusion ofcomponents of a material (e.g., an analyte of interest) into or withinthe fluidic medium. The rate of diffusion of components of such amaterial can depend on, for example, temperature, the size of thecomponents, and the strength of interactions between the components andthe fluidic medium.

As used herein in reference to different regions within a microfluidicdevice, the phrase “fluidically connected” means that, when thedifferent regions are substantially filled with fluid, such as fluidicmedia, the fluid in each of the regions is connected so as to form asingle body of fluid. This does not mean that the fluids (or fluidicmedia) in the different regions are necessarily identical incomposition. Rather, the fluids in different fluidically connectedregions of a microfluidic device can have different compositions (e.g.,different concentrations of solutes, such as proteins, carbohydrates,ions, or other molecules) which are in flux as solutes move down theirrespective concentration gradients and/or fluids flow through thedevice.

A microfluidic (or nanofluidic) device can comprise “swept” regions and“unswept” regions. As used herein, a “swept” region is comprised of oneor more fluidically interconnected circuit elements of a microfluidiccircuit, each of which experiences a flow of medium when fluid isflowing through the microfluidic circuit. The circuit elements of aswept region can include, for example, regions, channels, and all orparts of chambers. As used herein, an “unswept” region is comprised ofone or more fluidically interconnected circuit element of a microfluidiccircuit, each of which experiences substantially no flux of fluid whenfluid is flowing through the microfluidic circuit. An unswept region canbe fluidically connected to a swept region, provided the fluidicconnections are structured to enable diffusion but substantially no flowof media between the swept region and the unswept region. Themicrofluidic device can thus be structured to substantially isolate anunswept region from a flow of medium in a swept region, while enablingsubstantially only diffusive fluidic communication between the sweptregion and the unswept region. For example, a flow channel of amicro-fluidic device is an example of a swept region while an isolationregion (described in further detail below) of a microfluidic device isan example of an unswept region.

As used herein, a “flow path” refers to one or more fluidicallyconnected circuit elements (e.g. channel(s), region(s), chamber(s) andthe like) that define, and are subject to, the trajectory of a flow ofmedium. A flow path is thus an example of a swept region of amicrofluidic device. Other circuit elements (e.g., unswept regions) maybe fluidically connected with the circuit elements that comprise theflow path without being subject to the flow of medium in the flow path.

The capability of biological micro-objects (e.g., biological cells) toproduce specific biological materials (e.g., proteins, such asantibodies) can be assayed in such a microfluidic device. In a specificembodiment of an assay, sample material comprising biologicalmicro-objects (e.g., cells) to be assayed for production of an analyteof interest can be loaded into a swept region of the microfluidicdevice. Ones of the biological micro-objects (e.g., mammalian cells,such as human cells) can be selected for particular characteristics anddisposed in unswept regions. The remaining sample material can then beflowed out of the swept region and an assay material flowed into theswept region. Because the selected biological micro-objects are inunswept regions, the selected biological micro-objects are notsubstantially affected by the flowing out of the remaining samplematerial or the flowing in of the assay material. The selectedbiological micro-objects can be allowed to produce the analyte ofinterest, which can diffuse from the unswept regions into the sweptregion, where the analyte of interest can react with the assay materialto produce localized detectable reactions, each of which can becorrelated to a particular unswept region. Any unswept region associatedwith a detected reaction can be analyzed to determine which, if any, ofthe biological micro-objects in the unswept region are sufficientproducers of the analyte of interest.

Microfluidic Devices and Systems for Operating and Observing SuchDevices

FIG. 1 illustrates an example of a system 150 which can be used tocontrol a microfluidic device 100, such as a SSOEW device, in thepractice of the present invention. A perspective view of themicrofluidic device 100 is shown having a partial cut-away of a cover110 to provide a partial view into the microfluidic device 100. Ofcourse, the cover 110 may not always be present, or may be present onpart of the microfluidic device 100 but absent on another part of themicrofluidic device 100. The microfluidic device 100 generally comprisesa microfluidic circuit 120 comprising a flow path 106 through which afluidic medium 180 can flow, optionally carrying one or more droplets(not shown) and/or one or more micro-objects (not shown) into and/orthrough the microfluidic circuit 120. Although a single microfluidiccircuit 120 is illustrated in FIG. 1, suitable microfluidic devices caninclude a plurality (e.g., 2 or 3) of such microfluidic circuits.Regardless, the microfluidic device 100 can be configured to be ananofluidic device. In the embodiment illustrated in FIG. 1, themicrofluidic circuit 120 comprises a plurality of microfluidicsequestration pens 124, 126, 128, and 130, each having one or moreopenings in fluidic communication with flow path 106. As discussedfurther below, the microfluidic sequestration pens comprise variousfeatures and structures that have been optimized for sequestering andseparating droplets and/or micro-objects in the microfluidic device,such as microfluidic device 100, even when a medium 180 is flowingthrough the flow path 106.

As generally illustrated in FIG. 1, the microfluidic circuit 120 isdefined by an enclosure 102. Enclosure 102 is depicted as comprising asupport structure 104 (e.g., a base), a microfluidic circuit structure108, and a cover 110. However, enclosure 102 can be physicallystructured in different configurations; as alluded to above, themicrofluidic device 100 may lack a cover 110 over part or all of thesupport structure 104. Regardless, the support structure 104,microfluidic circuit structure 108, and cover 110 can be attached toeach other. For example, the microfluidic circuit structure 108 can bedisposed on an inner surface 109 of the support structure 104, and thecover 110 can be disposed over the microfluidic circuit structure 108.Together with the support structure 104 and cover 110, the microfluidiccircuit structure 108 can define the elements of the microfluidiccircuit 120. Alternatively, if the microfluidic device lacks a cover110, the microfluidic circuit structure 108 can be disposed on an innersurface 109 of the support structure 104, and together the supportstructure 104 and the microfluidic circuit structure 108 and can definethe elements of the microfluidic circuit 120.

There can be one or more ports 107 each comprising a passage into or outof the enclosure 102. Examples of a passage include a valve, a gate, apass-through hole, or the like. As illustrated, port 107 is apass-through hole created by a gap in the microfluidic circuit structure108. However, the port 107 can be situated in other components of theenclosure 102, such as the cover 110, if present. Only one port 107 isillustrated in FIG. 1 but the microfluidic circuit 120 can have two ormore ports 107. For example, there can be a first port 107 thatfunctions as an inlet for fluid entering the microfluidic circuit 120,and there can be a second port 107 that functions as an outlet for fluidexiting the microfluidic circuit 120. Whether a port 107 function as aninlet or an outlet can depend upon the direction that fluid flowsthrough flow path 106.

The support structure 104 can comprise one or more electrodes (notshown) and a substrate or a plurality of interconnected substrates. Forexample, the support structure 104 can comprise one or moresemiconductor substrates, each of which is electrically connected to oneor more electrodes (e.g., all or a subset of the semiconductorsubstrates can be electrically connected to a common electrode). Thesupport structure 104 can further comprise a printed circuit boardassembly (“PCBA”). For example, the semiconductor substrate(s) can bemounted on a PCBA.

The microfluidic circuit structure 108 can define circuit elements ofthe microfluidic circuit 120. Such circuit elements can comprise spacesor regions that can be fluidly interconnected when microfluidic circuit120 is filled with or otherwise contains fluid, such as flow channels,chambers, pens, traps, and the like. In the microfluidic circuit 120illustrated in FIG. 1, the microfluidic circuit structure 108 comprisesa frame 114 and a microfluidic circuit material 116. The frame 114 canpartially or completely enclose the microfluidic circuit material 116.The frame 114 can be, for example, a relatively rigid structuresubstantially surrounding the microfluidic circuit material 116. Forexample the frame 114 can comprise a metal material.

The microfluidic circuit material 116 can be patterned with cavities orthe like to define circuit elements and interconnections of themicrofluidic circuit 120. The microfluidic circuit material 116 cancomprise a flexible material, such as a flexible polymer (e.g. rubber,plastic, elastomer, silicone, polydimethylsiloxane (“PDMS”), or thelike), which can be gas permeable. Other examples of materials that cancompose microfluidic circuit material 116 include molded glass, anetchable material such as silicone (e.g. photo-patternable silicone),photo-resist (e.g., SU8), or the like. In some embodiments, suchmaterials—and thus the microfluidic circuit material 116—can be rigidand/or substantially impermeable to gas. Regardless, microfluidiccircuit material 116 can be disposed on the support structure 104 andinside the frame 114.

The cover 110, if present, can be an integral part of the frame 114and/or the microfluidic circuit material 116. Alternatively, the cover110 can be a structurally distinct element, as illustrated in FIG. 1.The cover 110 can comprise the same or different materials than theframe 114 and/or the microfluidic circuit material 116. Similarly, thesupport structure 104 can be a separate structure from the frame 114 ormicrofluidic circuit material 116 as illustrated, or an integral part ofthe frame 114 or microfluidic circuit material 116. Likewise the frame114 and microfluidic circuit material 116 can be separate structures asshown in FIG. 1 or integral portions of the same structure.

In some embodiments, the cover 110, if present, can comprise apierceable and/or deformable material. For example, the cover cancomprise a flexible polymer, such as rubber, plastic, elastomer,silicone, polydimethylsiloxane (“PDMS”), or the like. In otherembodiments, the cover 110 can comprise a rigid material. The rigidmaterial may be glass or a material with similar properties. In someembodiments, the cover 110 can comprise both rigid andpierceable/deformable materials. For example, one or more portions ofcover 110 (e.g., one or more portions positioned over sequestration pens124, 126, 128, 130) can comprise a deformable material that interfaceswith rigid materials of the cover 110. In some embodiments, the cover110, or portions of it, can further include one or more electrodes. Theone or more electrodes can comprise a conductive oxide, such asindium-tin-oxide (ITO), which may be coated on glass or a similarlyrigid insulating material. Alternatively, the one or more electrodes canbe flexible electrodes, such as single-walled nanotubes, multi-wallednanotubes, nanowires, clusters of electrically conductive nanoparticles,or combinations thereof, embedded in a deformable material, such as apolymer (e.g., PDMS). Flexible electrodes that can be used inmicrofluidic devices have been described, for example, in U.S.2012/0325665 (Chiou et al.), the contents of which are incorporatedherein by reference. In some embodiments, the cover 110 can be modified(e.g., by conditioning all or part of a surface that faces inward towardthe microfluidic circuit 120) to support cell adhesion, viability and/orgrowth. The modification may include a coating of a synthetic or naturalpolymer. In some embodiments, the cover 110 and/or the support structure104 can be transparent to light. The cover 110 may also include at leastone material that is gas permeable (e.g., PDMS or PPS).

FIG. 1 also shows a system 150 for operating and controllingmicrofluidic devices, such as microfluidic device 100. System 150, asillustrated, includes an electrical power source 192, an imaging device194, and a tilting device 190.

The electrical power source 192 can provide electric power to themicrofluidic device 100 and/or tilting device 190, providing biasingvoltages or currents as needed. The electrical power source 192 can, forexample, comprise one or more alternating current (AC) and/or directcurrent (DC) voltage or current sources. The imaging device 194 cancomprise a device, such as a digital camera, for capturing images insidemicrofluidic circuit 120. In some instances, the imaging device 194further comprises a detector having a fast frame rate and/or highsensitivity (e.g. for low light applications). The imaging device 194can also include a mechanism for directing stimulating radiation and/orlight beams into the microfluidic circuit 120 and collecting radiationand/or light beams reflected or emitted from the microfluidic circuit120 (or micro-objects contained therein). The emitted light beams may bein the visible spectrum and may, e.g., include fluorescent emissions.The reflected light beams may include reflected emissions originatingfrom an LED or a wide spectrum lamp, such as a mercury lamp (e.g. a highpressure mercury lamp) or a Xenon arc lamp. As discussed with respect toFIG. 3, the imaging device 194 may further include a microscope (or anoptical train), which may or may not include an eyepiece.

System 150 further comprises a tilting device 190 configured to rotate amicrofluidic device 100 about one or more axes of rotation. In someembodiments, the tilting device 190 is configured to support and/or holdthe enclosure 102 comprising the microfluidic circuit 120 about at leastone axis such that the microfluidic device 100 (and thus themicrofluidic circuit 120) can be held in a level orientation (i.e. at 0°relative to x- and y-axes), a vertical orientation (i.e. at 90° relativeto the x-axis and/or the y-axis), or any orientation therebetween. Theorientation of the microfluidic device 100 (and the microfluidic circuit120) relative to an axis is referred to herein as the “tilt” of themicrofluidic device 100 (and the microfluidic circuit 120). For example,the tilting device 190 can tilt the microfluidic device 100 at 0.1°,0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 10°,15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°,90° relative to the x-axis or any degree therebetween. The levelorientation (and thus the x- and y-axes) is defined as normal to avertical axis defined by the force of gravity. The tilting device canalso tilt the microfluidic device 100 (and the microfluidic circuit 120)to any degree greater than 90° relative to the x-axis and/or y-axis, ortilt the microfluidic device 100 (and the microfluidic circuit 120) 180°relative to the x-axis or the y-axis in order to fully invert themicrofluidic device 100 (and the microfluidic circuit 120). Similarly,in some embodiments, the tilting device 190 tilts the microfluidicdevice 100 (and the microfluidic circuit 120) about an axis of rotationdefined by flow path 106 or some other portion of microfluidic circuit120.

In some instances, the microfluidic device 100 is tilted into a verticalorientation such that the flow path 106 is positioned above or below oneor more sequestration pens. The term “above” as used herein denotes thatthe flow path 106 is positioned higher than the one or moresequestration pens on a vertical axis defined by the force of gravity(i.e. an object in a sequestration pen above a flow path 106 would havea higher gravitational potential energy than an object in the flowpath). The term “below” as used herein denotes that the flow path 106 ispositioned lower than the one or more sequestration pens on a verticalaxis defined by the force of gravity (i.e. an object in a sequestrationpen below a flow path 106 would have a lower gravitational potentialenergy than an object in the flow path).

In some instances, the tilting device 190 tilts the microfluidic device100 about an axis that is parallel to the flow path 106. Moreover, themicrofluidic device 100 can be tilted to an angle of less than 90° suchthat the flow path 106 is located above or below one or moresequestration pens without being located directly above or below thesequestration pens. In other instances, the tilting device 190 tilts themicrofluidic device 100 about an axis perpendicular to the flow path106. In still other instances, the tilting device 190 tilts themicrofluidic device 100 about an axis that is neither parallel norperpendicular to the flow path 106.

System 150 can further include a media source 178. The media source 178(e.g., a container, reservoir, or the like) can comprise multiplesections or containers, each for holding a different fluidic medium 180.Thus, the media source 178 can be a device that is outside of andseparate from the microfluidic device 100, as illustrated in FIG. 1.Alternatively, the media source 178 can be located in whole or in partinside the enclosure 102 of the microfluidic device 100. For example,the media source 178 can comprise reservoirs that are part of themicrofluidic device 100. The fluidic medium can be a hydrophobic medium,such as an oil (e.g., a silicone oil, a fluorinated oil, or acombination thereof), or a hydrophilic medium, such as a aqueoussolution (e.g., a saline solution or a cell culture medium).

FIG. 1 also illustrates simplified block diagram depictions of examplesof control and monitoring equipment 152 that constitute part of system150 and can be utilized in conjunction with a microfluidic device 100.As shown, examples of such control and monitoring equipment 152 includea master controller 154 comprising a media module 160 for controllingthe media source 178, a motive module 162 for controlling movementand/or selection of micro-objects (not shown) and/or medium (e.g.,droplets of medium) in the microfluidic circuit 120, an imaging module164 for controlling an imaging device 194 (e.g., a camera, microscope,light source or any combination thereof) for capturing images (e.g.,digital images), and a tilting module 166 for controlling a tiltingdevice 190. The control equipment 152 can also include other modules 168for controlling, monitoring, or performing other functions with respectto the microfluidic device 100. As shown, the equipment 152 can furtherinclude a display device 170 and an input/output device 172.

The master controller 154 can comprise a control module 156 and adigital memory 158. The control module 156 can comprise, for example, adigital processor configured to operate in accordance with machineexecutable instructions (e.g., software, firmware, source code, or thelike) stored as non-transitory data or signals in the memory 158.Alternatively or in addition, the control module 156 can comprisehardwired digital circuitry and/or analog circuitry. The media module160, motive module 162, imaging module 164, tilting module 166, and/orother modules 168 can be similarly configured. Thus, functions,processes acts, actions, or steps of a process discussed herein as beingperformed with respect to the microfluidic device 100 or any othermicrofluidic apparatus can be performed by any one or more of the mastercontroller 154, media module 160, motive module 162, imaging module 164,tilting module 166, and/or other modules 168 configured as discussedabove. Similarly, the master controller 154, media module 160, motivemodule 162, imaging module 164, tilting module 166, and/or other modules168 may be communicatively coupled to transmit and receive data used inany function, process, act, action or step discussed herein.

The media module 160 controls the media source 178. For example, themedia module 160 can control the media source 178 to input a selectedfluidic medium 180 into the enclosure 102 (e.g., through an inlet port107 or introduced directly into the enclosure 102). The media module 160can also control removal of media from the enclosure 102 (e.g., throughan outlet port (not shown) or direct removal). One or more media canthus be selectively input into and removed from the microfluidic circuit120. The media module 160 can also control the flow of fluidic medium180 in the flow path 106 inside the microfluidic circuit 120. Forexample, in some embodiments media module 160 stops the flow of media180 in the flow path 106 and through the enclosure 102 prior toperforming some other operation, such as using an opto-electrowettingforce or other type of force (e.g., dielectrophoresis or gravity) tomove a droplet and/or micro-object within the enclosure 102.

The motive module 162 can be configured to control selection, trapping,and movement of droplets and/or micro-objects (not shown) in themicrofluidic circuit 120. As discussed below, the enclosure 102 cancomprise an opto-electrowetting (OEW), dielectrophoresis (DEP), and/oroptoelectronic tweezers (OET) configuration (not shown in FIG. 1), andthe motive module 162 can control the activation of electrodes and/ortransistors (e.g., phototransistors) to select and move micro-objects(not shown) and/or droplets of medium (not shown) in the flow path 106and/or sequestration pens 124, 126, 128, 130.

The imaging module 164 can control the imaging device 194. For example,the imaging module 164 can receive and process image data from theimaging device 194. Image data from the imaging device 194 can compriseany type of information captured by the imaging device 194 (e.g., thepresence or absence of droplets of medium, micro-objects, or the like).Using the information captured by the imaging device 194, the imagingmodule 164 can further calculate the position of objects (e.g.,micro-objects, droplets of medium) and/or the rate of motion of suchobjects within the microfluidic device 100.

The tilting module 166 can control the tilting motions of tilting device190. Alternatively or in addition, the tilting module 166 can controlthe tilting rate and timing to optimize transfer of micro-objects to theone or more sequestration pens via gravitational forces. The tiltingmodule 166 is communicatively coupled with the imaging module 164 toreceive data describing the motion of micro-objects and/or droplets ofmedium in the microfluidic circuit 120. Using this data, the tiltingmodule 166 may adjust the tilt of the microfluidic circuit 120 in orderto adjust the rate at which micro-objects and/or droplets of medium movein the microfluidic circuit 120. The tilting module 166 may also usethis data to iteratively adjust the position of a micro-object and/ordroplet of medium in the microfluidic circuit 120.

In the example shown in FIG. 1, the microfluidic circuit 120 isillustrated as comprising a microfluidic channel 122 and sequestrationpens 124, 126, 128, 130. Each pen comprises an opening to channel 122,but otherwise is enclosed such that the pens can substantially isolatedroplets and/or micro-objects inside the pen from fluidic medium 180,droplets, and/or micro-objects in the flow path 106 of channel 122 or inother pens. In some instances, pens 124, 126, 128, 130 are configured tophysically corral one or more droplets and/or micro-objects within themicrofluidic circuit 120. Sequestration pens in accordance with thepresent invention can comprise various shapes, surfaces and featuresthat are optimized for use with OEW, DEP, OET, and/or gravitationalforces, as will be discussed below.

The microfluidic circuit 120 may comprise any number of microfluidicsequestration pens. Although five sequestration pens are shown,microfluidic circuit 120 may have fewer or more sequestration pens. Asshown, microfluidic sequestration pens 124, 126, 128, and 130 ofmicrofluidic circuit 120 each comprise differing features and shapeswhich may provide one or more benefits useful for the manipulation ofdroplets and/or micro-objects. In some embodiments, the microfluidiccircuit 120 comprises a plurality of identical microfluidicsequestration pens. In some embodiments, the microfluidic circuit 120comprises a plurality of microfluidic sequestration pens, wherein two ormore of the sequestration pens comprise differing structures and/orfeatures which provide differing benefits. For example, microfluidicsequestration pens in accordance with the present invention may beconfigured to isolate droplets for processing cells (e.g., for nucleicacid extraction and/or processing), isolated cells for cell culture,and/or isolate cellular components, such as nucleic acids, proteins, ormetabolites, for processing and/or amplification.

In the embodiment illustrated in FIG. 1, a single channel 122 and flowpath 106 is shown. However, other embodiments may contain multiplechannels 122, each configured to comprise a flow path 106. Themicrofluidic circuit 120 further comprises an inlet valve or port 107 influid communication with the flow path 106 and fluidic medium 180,whereby fluidic medium 180 can access channel 122 via the inlet port107. In some instances, the flow path 106 comprises a single path. Insome instances, the single path is arranged in a zigzag pattern wherebythe flow path 106 travels across the microfluidic device 100 two or moretimes in alternating directions.

In some instances, microfluidic circuit 120 comprises a plurality ofparallel channels 122 and flow paths 106, wherein the fluidic medium 180within each flow path 106 flows in the same direction. In someinstances, the fluidic medium within each flow path 106 flows in atleast one of a forward or reverse direction. In some instances, aplurality of sequestration pens are configured (e.g., relative to achannel 122) such that they can be loaded with droplets and/or targetmicro-objects (e.g., cells) in parallel.

In some embodiments, microfluidic circuit 120 further comprises one ormore micro-object traps 132. The traps 132 are generally formed in awall forming the boundary of a channel 122, and may be positionedopposite an opening of one or more of the microfluidic sequestrationpens 124, 126, 128, 130. In some embodiments, the traps 132 areconfigured to receive or capture a single droplet or micro-object fromthe flow path 106. In some embodiments, the traps 132 are configured toreceive or capture a plurality of droplets or micro-objects from theflow path 106. In some instances, the traps 132 comprise a volumeapproximately equal to the volume of a single droplet or targetmicro-object.

The traps 132 may further comprise an opening 183 which is configured toassist the flow of droplets or targeted micro-objects into the traps132. In some instances, the traps 132 comprise an opening having a width(and, optionally, a height) that is approximately equal to thedimensions of a single target micro-object, whereby larger droplets ormicro-objects are prevented from entering into the micro-object trap.The traps 132 may further comprise other features configured to assistin retention of droplets or targeted micro-objects within the trap 132.In some instances, the trap 132 is aligned with and situated on theopposite side of a channel 122 relative to the opening of a microfluidicsequestration pen, such that upon tilting the microfluidic device 100about an axis parallel to the channel 122, the trapped droplet ormicro-object exits the trap 132 at a trajectory that causes themicro-object to fall into the opening of the sequestration pen. In someinstances, the trap 132 comprises a side passage 134 that is smallerthan the target micro-object in order to facilitate flow through thetrap 132 and thereby increase the likelihood of capturing a micro-objectin the trap 132.

In some embodiments, optoelectrowetting (OEW) forces are applied to oneor more positions in the support structure 104 (and/or the cover 110) ofthe microfluidic device 100 (e.g., positions helping to define the flowpath and/or the sequestration pens) via one or more electrodes (notshown) to manipulate, transport, separate and sort droplets located inthe microfluidic circuit 120. For example, in some embodiments, OEWforces are applied to one or more positions in the support structure 104(and/or the cover 110) in order to transfer a single droplet from theflow path 106 into a desired microfluidic sequestration pen. In someembodiments, OEW forces are used to prevent a droplet within asequestration pen (e.g., sequestration pen 124, 126, 128, or 130) frombeing displaced therefrom. Further, in some embodiments, OEW forces areused to selectively remove a droplet from a sequestration pen that waspreviously collected in accordance with the teachings of the instantinvention.

In other embodiments, dielectrophoretic (DEP) forces are applied acrossthe fluidic medium 180 (e.g., in the flow path and/or in thesequestration pens) via one or more electrodes (not shown) tomanipulate, transport, separate and sort micro-objects located therein.For example, in some embodiments, DEP forces are applied to one or moreportions of microfluidic circuit 120 in order to transfer a singlemicro-object from the flow path 106 into a desired microfluidicsequestration pen. In some embodiments, DEP forces are used to prevent amicro-object within a sequestration pen (e.g., sequestration pen 124,126, 128, or 130) from being displaced therefrom. Further, in someembodiments, DEP forces are used to selectively remove a micro-objectfrom a sequestration pen that was previously collected in accordancewith the teachings of the instant invention. In some embodiments, theDEP forces comprise optoelectronic tweezer (OET) forces.

In some embodiments, DEP and/or OEW forces are combined with otherforces, such as flow and/or gravitational force, so as to manipulate,transport, separate and sort micro-objects and/or droplets within themicrofluidic circuit 120. For example, the enclosure 102 can be tilted(e.g., by tilting device 190) to position the flow path 106 andmicro-objects located therein above the microfluidic sequestration pens,and the force of gravity can transport the micro-objects and/or dropletsinto the pens. In some embodiments, the DEP and/or OEW forces can beapplied prior to the other forces. In other embodiments, the DEP and/orOEW forces can be applied after the other forces. In still otherinstances, the DEP and/or OEW forces can be applied at the same time asthe other forces or in an alternating manner with the other forces.

A variety of optically-actuated electrokinetic devices are known in theart, including devices having an optoelectronic tweezer (OET)configuration and devices having an opto-electrowetting (OEW)configuration. Examples of OET configurations are illustrated in thefollowing U.S. patent documents, each of which is incorporated herein byreference in its entirety: U.S. Pat. No. RE 44,711 (Wu et al.)(originally issued as U.S. Pat. No. 7,612,355); and U.S. Pat. No.7,956,339 (Ohta et al.). Examples of OEW configurations are illustratedin U.S. Pat. No. 6,958,132 (Chiou et al.) and U.S. Patent ApplicationPublication No. 2012/0024708 (Chiou et al.), both of which areincorporated by reference herein in their entirety. Yet another exampleof an optically-actuated electrokinetic device includes a combinedOET/OEW configuration, examples of which are shown in U.S. PatentPublication Nos. 20150306598 (Khandros et al.) and 20150306599 (Khandroset al.) and their corresponding PCT Publications WO2015/164846 andWO2015/164847, all of which are incorporated herein by reference intheir entirety.

FIGS. 2A through 2D show various embodiments of system 150 which can beused to operate and observe microfluidic devices (e.g. 100) according tothe present invention. As illustrated in FIG. 2A, the system 150 caninclude a structure (“nest”) 200 configured to hold and operativelycouple with a microfluidic device 250, which can be a microfluidicdevice described herein. The nest 200 can include a socket 202 capableof interfacing with the microfluidic device 250 (e.g., anoptically-actuated electrokinetic device, such as a microfluidic devicehaving a SSOEW configuration) and providing electrical connections frompower source 192 to microfluidic device 250. The nest 200 can furtherinclude an integrated electrical signal generation subsystem 204. Theelectrical signal generation subsystem 204 can be configured to supply abiasing voltage to socket 202 such that the biasing voltage is appliedacross a pair of electrodes in the microfluidic device 250 when it isbeing held by socket 202. Thus, the electrical signal generationsubsystem 204 can be part of power source 192. The ability to apply abiasing voltage to microfluidic device 250 does not mean that a biasingvoltage will be applied at all times when the microfluidic device 250 isheld by the socket 202. Rather, in most cases, the biasing voltage willbe applied intermittently, e.g., only as needed to facilitate thegeneration of electrokinetic forces, such as electro-wetting ordielectrophoresis, in the microfluidic device 250.

As illustrated in FIG. 2A, the nest 200 can include a printed circuitboard assembly (PCBA) 220. The electrical signal generation subsystem204 can be mounted on and electrically integrated into the PCBA 220. Theexemplary support includes socket 202 mounted on PCBA 320, as well.

Typically, the electrical signal generation subsystem 204 will include awaveform generator (not shown). The electrical signal generationsubsystem 204 can further include an oscilloscope (not shown) and/or awaveform amplification circuit (not shown) configured to amplify awaveform received from the waveform generator. The oscilloscope, ifpresent, can be configured to measure the waveform supplied to themicrofluidic device 250 held by the socket 202. In certain embodiments,the oscilloscope measures the waveform at a location proximal to themicrofluidic device 250 (and distal to the waveform generator), thusensuring greater accuracy in measuring the waveform actually applied tothe device. Data obtained from the oscilloscope measurement can be, forexample, provided as feedback to the waveform generator, and thewaveform generator can be configured to adjust its output based on suchfeedback. An example of a suitable combined waveform generator andoscilloscope is the Red Pitaya™.

In certain embodiments, the nest 200 further comprises a controller 208,such as a microprocessor used to sense and/or control the electricalsignal generation subsystem 204. Examples of suitable microprocessorsinclude the Arduino™ microprocessors, such as the Arduino Nano™. Thecontroller 208 may be used to perform functions and analysis or maycommunicate with an external master controller 154 (shown in FIG. 1) toperform functions and analysis. In the embodiment illustrated in FIG. 2Athe controller 208 communicates with a master controller 154 through aninterface 210 (e.g., a plug or connector).

In some embodiments, the nest 200 can comprise an electrical signalgeneration subsystem 204 comprising a Red Pitaya™ waveformgenerator/oscilloscope unit (“Red Pitaya™ unit”) and a waveformamplification circuit that amplifies the waveform generated by the RedPitaya™ unit and passes the amplified voltage to the microfluidic device100. In some embodiments, the Red Pitaya™ unit is configured to measurethe amplified voltage at the microfluidic device 250 and then adjust itsown output voltage as needed such that the measured voltage at themicrofluidic device 250 is the desired value. In some embodiments, thewaveform amplification circuit can have a +6.5V to −6.5V power supplygenerated by a pair of DC-DC converters mounted on the PCBA 220,resulting in a signal of up to 13 Vpp at the microfluidic device 250.

As illustrated in FIG. 2A, the nest 200 can further include a thermalcontrol subsystem 206. The thermal control subsystem 206 can beconfigured to regulate the temperature of microfluidic device 250 heldby the nest 200. For example, the thermal control subsystem 206 caninclude a Peltier thermoelectric device (not shown) and a cooling unit(not shown). The Peltier thermoelectric device can have a first surfaceconfigured to interface with at least one surface of the microfluidicdevice 250. The cooling unit can be, for example, a cooling block (notshown), such as a liquid-cooled aluminum block. A second surface of thePeltier thermoelectric device (e.g., a surface opposite the firstsurface) can be configured to interface with a surface of such a coolingblock. The cooling block can be connected to a fluidic path 230configured to circulate cooled fluid through the cooling block. In theembodiment illustrated in FIG. 2A, the nest 200 comprises an inlet 232and an outlet 234 to receive cooled fluid from an external reservoir(not shown), introduce the cooled fluid into the fluidic path 230 andthrough the cooling block, and then return the cooled fluid to theexternal reservoir. In some embodiments, the Peltier thermoelectricdevice, the cooling unit, and/or the fluidic path 230 can be mounted ona casing 240 of the nest 200. In some embodiments, the thermal controlsubsystem 206 is configured to regulate the temperature of the Peltierthermoelectric device so as to achieve a target temperature for themicrofluidic device 250. Temperature regulation of the Peltierthermoelectric device can be achieved, for example, by a thermoelectricpower supply, such as a Pololu™ thermoelectric power supply (PololuRobotics and Electronics Corp.). The thermal control subsystem 206 caninclude a feedback circuit, such as a temperature value provided by ananalog circuit. Alternatively, the feedback circuit can be provided by adigital circuit.

In some embodiments, the nest 200 can include a thermal controlsubsystem 206 with a feedback circuit that is an analog voltage dividercircuit which includes a resistor (e.g., with resistance 1 kOhm+/−0.1%,temperature coefficient+/−0.02 ppm/C0) and a NTC thermistor (e.g., withnominal resistance 1 kOhm+/−0.01%). In some instances, the thermalcontrol subsystem 206 measures the voltage from the feedback circuit andthen uses the calculated temperature value as input to an on-board PIDcontrol loop algorithm. Output from the PID control loop algorithm candrive, for example, both a directional and a pulse-width-modulatedsignal pin on a Pololu™ motor drive (not shown) to actuate thethermoelectric power supply, thereby controlling the Peltierthermoelectric device.

The nest 200 can include a serial port 260 which allows themicroprocessor of the controller 208 to communicate with an externalmaster controller 154 via the interface 210. In addition, themicroprocessor of the controller 208 can communicate (e.g., via a Plinktool (not shown)) with the electrical signal generation subsystem 204and thermal control subsystem 206. Thus, via the combination of thecontroller 208, the interface 210, and the serial port 260, theelectrical signal generation subsystem 208 and the thermal controlsubsystem 206 can communicate with the external master controller 154.In this manner, the master controller 154 can, among other things,assist the electrical signal generation subsystem 208 by performingscaling calculations for output voltage adjustments. A Graphical UserInterface (GUI) provided via a display device 170 coupled to theexternal master controller 154, can be configured to plot temperatureand waveform data obtained from the thermal control subsystem 206 andthe electrical signal generation subsystem 208, respectively.Alternatively, or in addition, the GUI can allow for updates to thecontroller 208, the thermal control subsystem 206, and the electricalsignal generation subsystem 204.

As discussed above, system 150 can include an imaging device 194. Insome embodiments, the imaging device 194 comprises a light modulatingsubsystem 304. The light modulating subsystem 304 can include a digitalmirror device (DMD) or a microshutter array system (MSA), either ofwhich can be configured to receive light from a light source 302 andtransmits a subset of the received light into an optical train ofmicroscope 300. Alternatively, the light modulating subsystem 304 caninclude a device that produces its own light (and thus dispenses withthe need for a light source 302), such as an organic light emittingdiode display (OLED), a liquid crystal on silicon (LCOS) device, aferroelectric liquid crystal on silicon device (FLCOS), or atransmissive liquid crystal display (LCD). The light modulatingsubsystem 304 can be, for example, a projector. Thus, the lightmodulating subsystem 304 can be capable of emitting both structured andunstructured light. One example of a suitable light modulating subsystem304 is the Mosaic™ system from Andor Technologies™. In certainembodiments, imaging module 164 and/or motive module 162 of system 150can control the light modulating subsystem 304.

In certain embodiments, the imaging device 194 further comprises amicroscope 300. In such embodiments, the nest 200 and light modulatingsubsystem 304 can be individually configured to be mounted on themicroscope 300. The microscope 300 can be, for example, a lightmicroscope or fluorescence microscope. Thus, the nest 200 can beconfigured to be mounted on the stage 310 of the microscope 300 and/orthe light modulating subsystem 304 can be configured to mount on a portof microscope 300. In other embodiments, the nest 200 and the lightmodulating subsystem 304 described herein can be integral components ofmicroscope 300.

In certain embodiments, the microscope 300 can further include one ormore detectors 322. In some embodiments, the detector 422 is controlledby the imaging module 164. The detector 322 can include an eye piece, acharge-coupled device (CCD), a camera (e.g., a digital camera), or anycombination thereof. If at least two detectors 322 are present, onedetector can be, for example, a fast-frame-rate camera while the otherdetector can be a high sensitivity camera. Furthermore, the microscope300 can include an optical train configured to receive reflected and/oremitted light from the microfluidic device 250 and focus at least aportion of the reflected and/or emitted light on the one or moredetectors 322. The optical train of the microscope can also includedifferent tube lenses (not shown) for the different detectors, such thatthe final magnification on each detector can be different.

In certain embodiments, imaging device 194 is configured to use at leasttwo light sources. For example, a first light source 302 can be used toproduce structured light (e.g., via the light modulating subsystem 304)and a second light source 332 can be used to provide unstructured light.The first light source 302 can produce structured light foroptically-actuated electrokinesis and/or fluorescent excitation, and thesecond light source 332 can be used to provide bright fieldillumination. In these embodiments, the motive module 162 can be used tocontrol the first light source 304 and the imaging module 164 can beused to control the second light source 332. The optical train of themicroscope 300 can be configured to (1) receive structured light fromthe light modulating subsystem 304 and focus the structured light on atleast a first region in a microfluidic device, such as anoptically-actuated electrokinetic device (e.g., a microfluidic devicehaving a SSOEW configuration), when the device is being held by the nest200, and (2) receive reflected and/or emitted light from themicrofluidic device and focus at least a portion of such reflectedand/or emitted light onto detector 322. The optical train can be furtherconfigured to receive unstructured light from a second light source andfocus the unstructured light on at least a second region of themicrofluidic device, when the device is held by the nest 200. In certainembodiments, the first and second regions of the microfluidic device canbe overlapping regions. For example, the first region can be a subset ofthe second region.

In FIG. 2B, the first light source 302 is shown supplying light to alight modulating subsystem 304, which provides structured light to theoptical train of the microscope 300. The second light source 332 isshown providing unstructured light to the optical train via a beamsplitter 336. Structured light from the light modulating subsystem 304and unstructured light from the second light source 332 travel from thebeam splitter 336 through the optical train together to reach a secondbeam splitter 336 (or dichroic filter 306 depending on the lightprovided by the light modulating subsystem 304), where the light getsreflected down through the objective 308 to the sample plane 312.Reflected and/or emitted light from the sample plane 312 then travelsback up through the objective 308, through the beam splitter/dichroicfilter 306, and to a dichroic filter 324. Only a fraction of the lightreaching dichroic filter 324 passes through and reaches the detector322.

In some embodiments, the second light source 332 emits blue light. Withan appropriate dichroic filter 324, blue light reflected from the sampleplane 312 is able to pass through dichroic filter 324 and reach thedetector 322. In contrast, structured light coming from the lightmodulating subsystem 304 gets reflected from the sample plane 312, butdoes not pass through the dichroic filter 324. In this example, thedichroic filter 324 is filtering out visible light having a wavelengthlonger than 495 nm. Such filtering out of the light from the lightmodulating subsystem 304 would only be complete (as shown) if the lightemitted from the light modulating subsystem did not include anywavelengths shorter than 495 nm. In practice, if the light coming fromthe light modulating subsystem 304 includes wavelengths shorter than 495nm (e.g., blue wavelengths), then some of the light from the lightmodulating subsystem would pass through filter 324 to reach the detector322. In such an embodiment, the filter 324 acts to change the balancebetween the amount of light that reaches the detector 322 from the firstlight source 302 and the second light source 332. This can be beneficialif the first light source 302 is significantly stronger than the secondlight source 332. In other embodiments, the second light source 332 canemit red light, and the dichroic filter 324 can filter out visible lightother than red light (e.g., visible light having a wavelength shorterthan 650 nm).

Substrates Having a Single-Sided Optoelectrowetting (SSOEW)Configuration.

In some embodiments, a SSOEW substrate of the invention can include aplanar electrode, a photoconductive (or photosensitive) layer, adielectric layer, a mesh electrode, and a hydrophobic coating. Anexample of a SSOEW substrate 500 of the invention is shown in FIGS.3A-B. The photoconductive layer 504 can be interposed between the planarelectrode 502 and the dielectric layer 506; and the mesh electrode 508can adjoin a top surface of the dielectric layer (i.e., the surfaceopposite to the side that contacts the photoconductive layer). Theinsulating coating 510 can cover the dielectric layer 506, as well asany part of the mesh electrode 508 that is not embedded in thedielectric layer 506.

The dielectric layer 506 can be a single layer, as depicted in FIGS.3A-B, and can comprise, consist essentially of, or consist of an oxide.The oxide can have a dielectric constant of about 5 to about 15 (e.g.,about 7.5 to about 12.5, or about 9 to about 11.5). As an example, theoxide can be a metal oxide, such as aluminum oxide or hafnium oxide. Thedielectric layer 506 can be formed, for example, by atomic layerdeposition (ALD). Use of ALD for the formation of the dielectric layer506 (or portions thereof, as discussed further below) can beadvantageous because it deposits conformal films, with well-controlledthickness, that are substantially pinhole free (i.e., there are few tonone electrical shorts through the dielectric layer).

Alternatively, the dielectric layer 506 can have a multi-layer (or“composite”) structure that includes 2, 3, or more layers, each of whichcomprises a dielectric material. For example, as shown in FIGS. 5A-B and6C, the dielectric layer 506 can have a first dielectric layer 506 a anda second dielectric layer 506 b, with the bottom surface of the firstdielectric layer 506 a adjoining the photoconductive layer 504. The meshelectrode can be interposed between the first dielectric layer 506 a andthe second dielectric layer 506 b of a two-layer composite dielectriclayer 506, as shown. However, this is not a requirement, as the meshelectrode can rest upon (e.g., be adjacent to) the top surface of thesecond dielectric layer 506 b.

The first dielectric layer 506 a and second dielectric layer 506 b of atwo-layer composite dielectric layer 506 can include similar orsubstantially identical dielectric materials. For example, both thefirst dielectric layer 506 a and the second dielectric layer 506 b cancomprise a metal oxide, such as aluminum oxide or hafnium oxide. Inaddition, one or both of the first dielectric layer 506 a and the seconddielectric layer 506 b can be formed by ALD. When the compositedielectric layer 506 is configured in this manner, the first dielectriclayer 506 a can be a larger (e.g., thicker) component of the compositedielectric layer 506. For example, the first dielectric layer 506 a canhave a thickness of at least about 100 nm, at least about 125 nm, atleast about 150 nm, or more (or about 125 nm to about 175 nm, or about140 nm to about 160 nm), and the second dielectric layer 506 b can havea thickness of about 50 nm or less (e.g., about 40 nm or less, about 30nm or less, about 25 nm or less, about 20 nm or less, about 15 nm orless, about 10 nm or less, about 9 nm or less, about 8 nm or less, about7 nm or less, about 6 nm or less, about 5 nm or less, about 4 nm orless, or about 3 nm or less). In particular, the first dielectric layer506 a can have a thickness of at least about 125 nm (e.g., at leastabout 150 nm) and the second dielectric layer 506 b can have a thicknessof about 10 nm or less (e.g., about 5 nm or less).

The first dielectric layer 506 a of a composite dielectric layer 506 canhave a lattice shape, as shown in FIG. 6C (in cross-section). Thelattice can be of substantially uniform thickness, with the top surfaceof the first dielectric layer 506 a adjoining a bottom surface of themesh electrode 508 and the mesh electrode 508 interposed between thefirst dielectric layer 506 a and the second dielectric layer 506 b. Thetop surface of the first dielectric layer 506 a can be substantiallycontiguous with the bottom surface of the mesh electrode 508 (i.e., thewidth of individual linear elements of the lattice of the firstdielectric layer 506 a can be substantially equal to the width ofindividual wires in the mesh electrode, as shown in FIG. 6C). Configuredin this manner, the first dielectric layer 506 a and the seconddielectric layer 506 b of the composite dielectric layer 506 can includedifferent dielectric materials. For example, the first dielectric layer506 a can include a metal oxide, such as aluminum oxide or hafniumoxide, and the second dielectric layer 506 b can include a non-metaloxide, such as silicon oxide. The thickness of the first dielectriclayer 506 a can be at least about 50 nm, at least about 75 nm, at leastabout 100 nm, at least about 125 nm, at least about 150 nm, or more. Forexample, the first dielectric layer 506 a can have a thickness of about50 nm to about 200 nm, about 75 nm to about 180 nm, about 100 nm toabout 160 nm, or about 125 nm to about 150 nm. And the thickness of thesecond dielectric layer 506 b can be variable. For example, the seconddielectric layer 506 b can have first regions contacting the top surfaceof the photoconductive layer 504 with a thickness substantially the sameas the thickness of the composite dielectric layer 506, and secondregions directly over the wires of the mesh electrode 508 having athickness of about 100 nm or less (e.g., about 75 nm or less, about 50nm or less, about 40 nm or less, about 30 nm or less, about 25 nm orless, about 20 nm or less, about 15 nm or less, about 10 nm or less,about 9 nm or less, about 8 nm or less, about 7 nm or less, about 6 nmor less, about 5 nm or less, about 4 nm or less, or about 3 nm or less).In particular, the first dielectric layer 506 a can include a metaloxide and have a thickness of at least about 125 nm (e.g., at leastabout 150 nm), and the second dielectric layer 506 b can include anon-metal oxide and have a thickness directly over the wires of the meshelectrode 508 of about 10 nm or less (e.g., about 5 nm or less).

The first dielectric layer 506 a of a composite dielectric layer 506 cancomprise, consist essentially of, or consist of a first material havinga dielectric constant and the second dielectric layer 506 b cancomprise, consist essentially of, or consist of a second material havinga dielectric constant ∈₂, where ∈₁ is different than ∈₂ (e.g., ∈₁ can beless than ∈₂). For example, the first material can be a metal oxide,such as aluminum oxide or hafnium oxide, and the second material can bea non-metal oxide, such as silicon oxide.

As shown in FIG. 6A, a composite dielectric layer 506 can have a firstdielectric layer 506 a, a second dielectric layer 506 b, and a thirddielectric layer 506 c, with the bottom surface of the first dielectriclayer 506 a adjoining the photoconductive layer 504 and the seconddielectric layer 506 b interposed between the first dielectric layer 506a and the third dielectric layer 506 c. Configured in this manner, thefirst dielectric layer 506 a and the third dielectric layer 506 c caninclude similar or substantially identical dielectric materials. Forexample, the first dielectric layer 506 a and the third dielectric layer506 c can each comprise, consist essentially of, or consist of a metaloxide (e.g., aluminum oxide or hafnium oxide). In addition, the firstdielectric layer 506 a and the third dielectric layer 506 c can beformed, for example, by ALD. Alternatively, the first dielectric layer506 a and the third dielectric layer 506 c can include differentdielectric materials and/or can be formed by different processes.

The second dielectric layer 506 b of a three-layer composite dielectriclayer 506 can include a dielectric material that is different from thatof the first dielectric layer 506 a and/or the third dielectric layer506 c. The second dielectric layer 506 b can comprise, consistessentially of, or consist of, for example, a non-metal oxide, such assilicon oxide, or a nitride. In addition, the second dielectric layer506 b can be formed in a manner different from that of the firstdielectric layer 506 a and/or the third dielectric layer 506 c. Forexample, the second dielectric layer 506 b can be formed by plasmaenhanced chemical vapor deposition.

For a three-layer composite dielectric layer 506, each of the firstdielectric layer 506 a and the third dielectric layer 506 c can have athickness of at least about 5 nm (e.g., about 6 nm to about 12 nm, about7 nm to about 14 nm, about 8 nm to about 16 nm, about 9 nm to about 18nm, or about 10 nm to about 20 nm). The second dielectric layer 506 bcan have a thickness of at least about 75 nm (e.g., about 100 nm toabout 300 nm, about 110 nm to about 275 nm, about 120 nm to about 250nm, about 130 nm to about 225 nm, or about 140 nm to about 200 nm).

The first dielectric layer 506 a of a three-layer composite dielectriclayer 506 can have a top surface adjoining a bottom surface of the meshelectrode 508, and the third dielectric layer 506 c can have a bottomsurface adjoining a top surface of the mesh electrode 508. Thus, forexample, the mesh ground electrode can be entirely encased within thecomposite dielectric layer, with the second dielectric layer 506 bfilling the spaces formed between the lateral edges of the wires of themesh ground electrode. Alternatively, the mesh electrode 508 can restupon (e.g., be adjacent to) the top surface of the third dielectriclayer 506 c.

The first dielectric layer 506 a of a three-layer composite dielectriclayer 506 can be made from a first material that has a dielectricconstant the second dielectric layer 506 b can be made from a secondmaterial that has a dielectric constant ∈₂, and the third dielectriclayer 506 c can be made from a third material that has a dielectricconstant ∈₃. Depending on the configuration of the mesh electrode 508relative to the layers of the composite dielectric layer 506, ∈₁ can bedifferent than ∈₃. For example, ∈₁ can be similar to or substantiallythe same as ∈₃, and ∈₂ can be greater than ∈₁ and ∈₃. Alternatively, ∈₁can be less than ∈₃, and ∈₂ can be less than ∈₃ (e.g., ∈₂ can have avalue equal to or greater than ∈₁ but less than ∈₃).

A single-layer dielectric layer 506 can have a thickness of at least 50nm, at least about 75 nm, at least about 100 nm, at least about 125 nm,at least about 150 nm, or more. Thus, the dielectric layer 506 can havea thickness ranging from about 50 to about 250 nm, about 75 nm to about225 nm, about 100 nm to about 200 nm, about 125 nm to about 175 nm, orabout 140 nm to about 160 nm. Similarly, a composite dielectric layer(two-layer, three-layer, or more) can have an overall thickness of atleast about 50 nm, at least about 75 nm, at least about 100 nm, at leastabout 125 nm, at least about 150 nm, or more. For example, the compositedielectric layer can have an overall thickness of about 50 to about 250nm, about 75 nm to about 225 nm, about 100 nm to about 200 nm, about 125nm to about 175 nm, or about 140 nm to 160 nm.

As one skilled in the art will understand, the number of layers in thedielectric layer 506 and the overall thickness of the dielectric layer506 can be varied, depending upon other elements and design features ofthe SSOEW-configured substrate 500, such that the electrical impedanceof the dielectric layer 506 is suitable for achieving the desiredeffect—an SSOEW-configured substrate that can reliably and controllablyproduce an EW force. Generally, the dielectric layer 506 (whethersingle-layer or multi-layer) will have an electrical impedance of about10 kOhms to about 50 kOhms, or about 10 kOhms to about 20 kOhms. Forexample, the dielectric layer 506 can have an overall thickness of atleast 125 nm (e.g., at least 150 nm), inclusive of all of the one ormore dielectric layers (e.g., 506 a, 506 b, 506 c, etc.), and anelectrical impedance of about 10 kOhms to about 50 kOhms (e.g., about 10kOhms to about 20 kOhms).

As illustrated in FIGS. 3A and 5A, the mesh electrode 508 can comprise aplurality of wires arranged in a lattice shape. The wires of the meshelectrode 508 can have a substantially square shape or a substantiallyrectangular shape, when viewed in cross-section (see FIGS. 3B, 4A, 5B,and 6C). Thus, the wires can have an average width and an averageheight. The average height of the wires can be at least about 50 nm, atleast about 60 nm, at least about 70 nm, at least about 80 nm, at leastabout 90 nm, or at least about 100 nm. The average width of the wirescan be at least about 100 nm, at least about 200 nm, at least about 300nm, at least about 400 nm, at least about 500 nm, at least about 600 nm,at least about 700 nm, at least about 800 nm, at least about 900 nm, orat least about 1000 nm. However, other shapes are also possible and havethe potential to improve the performance of the SSOEW-configuredsubstrate 500. For example, the wires of the mesh electrode 508 can havea T-shape when viewed in cross section (see FIG. 6A). In addition, themesh electrode 508 can further comprise plates located on top of thevertices formed by the wires of the mesh electrode 508 (see FIG. 6B).The plates can have substantially the same composition as the wires ofthe mesh ground electrode 508. The wires of the mesh electrode 508 canbe directly deposited on the dielectric layer 506 (or a first dielectriclayer 506 a, a second dielectric layer 506 b, or a third dielectriclayer 506 c, etc., of a composite dielectric layer 506), and can be ofsubstantially uniform thickness.

The wires of the mesh electrode 508 can comprise (or consist essentiallyof, or consist of) a conductive material, such as a metal, a metalalloy, or a highly electrically conductive semiconductor material. Forexample, the wires of the mesh electrode 508 can comprise gold,aluminum, titanium, chromium, combinations thereof, and/or oxidizedvariants thereof. In particular, if the wires of the mesh electrodecomprise gold, they can include a layer of chromium or titanium (e.g.,to help bind the gold to the underlying dielectric layer 506).Alternatively, the wires of the mesh electrode 508 can comprisehighly-doped silicon. Optionally, the conductive material used in themesh electrode 508 can be non-toxic to biological (e.g., animalian,mammalian, or human) cells.

The mesh electrode 508 can have a linear fill factor β that is less thanor equal to about 10% (e.g., less than about 9%, less than about 8%,less than about 7%, less than about 6%, less than about 5%, less thanabout 4%, less than about 3%, less than about 2%, less than about 1%, orless than about 0.5%). The wires of the mesh electrode 508 can have apitch of less than 1 mm. For example, the wires of the mesh groundelectrode 508 can have a pitch of about 1.5 mm to about 2 mm, about 1.0mm to about 1.5 mm, about 0.5 mm to about 1.0 mm, about 400 to about 800microns, about 300 to about 600 microns, about 200 to about 400 microns,about 100 to about 200 microns, about 50 to about 100 microns, or about10 to about 50 microns. As discussed further below, the pitch of thewires can be adjusted depending on the size of the droplets that will bemoved upon the SSOEW-configured substrate 500.

As illustrated in FIGS. 3A-B, 4A, 5A-B, 6A, and 6C, the hydrophobiccoating 510 of a SSOEW-configured substrate 500 has a bottom surfacethat adjoins some or all of the top surface of the dielectric layer 506.Provided that the mesh electrode 508 rests on top of the dielectriclayer 506, and is not embedded in the dielectric layer 506 (e.g., asshown in FIGS. 3A-B and 4A), the bottom surface of the hydrophobiccoating 510 will also adjoin the top surfaces (and possibly the lateralsurfaces) of the wires of the mesh electrode 508. To achieve appropriatebonding between the hydrophobic coating 510 and exposed surfaces on thewires of the mesh electrode 508, the exposed surfaces of the wires mayrequire conditioning. For example, gold tends to bond poorly to themolecules of the hydrophobic coating 510, but this can be overcome bypassivating any surfaces of the gold that will contact the hydrophobiccoating 510. Such passivation can be achieve, for example, by reactingthe gold surfaces with thiol-containing molecules. Similarly, if thewires of the mesh electrode 508 comprise aluminum, surfaces that willcontact the hydrophobic coating 510 can be passivated. Aluminumpassivation can be achieved, for example, by surface oxidation (e.g., byplasma treatment of the exposed surfaces of the aluminum wires in anoxygen plasma chamber).

The hydrophobic coating 510 can comprise an organofluorine polymer,which can optionally include at least one perfluorinated segment. Forexample, the organofluorine polymer can comprisepolytetrafluoro-ethylene (PTFE) (i.e., Teflon®) orpoly(2,3-difluoromethylenyl-perfluorotetrahydrofuran) (i.e., Cytop™).The chemical structure ofpoly(2,3-difluoromethylenyl-perfluorotetrahydrofuran) is as follows:

Hydrophobic coatings 510 that comprise an organofluorine polymer, suchas polytetrafluoro-ethylene (PTFE) orpoly(2,3-difluoromethylenyl-perfluorotetrahydrofuran), can have athickness of at least about 10 nm, at least about 15 nm, at least about20 nm, at least about 25 nm, at least about 30 nm, or at least about 35nm. For example, the hydrophobic coating 510 can have a thickness ofabout 25 nm to about 40 nm.

The hydrophobic layer 510 can comprise a densely packed monolayer ofamphiphilic molecules covalently bonded to molecules of the top surfaceof the dielectric layer 506 (and/or surfaces of the mesh electrode 508that are not embedded in the dielectric layer 506). Such amphiphilicmolecules can each comprise a siloxane group, a phosphonic acid group,or a thiol group, and the respective siloxane groups, phosphonic acidgroups, and thiol groups can form the covalent bonds with the moleculesof the dielectric layer 506. As used herein, a “densely packedmonolayer” of amphiphilic molecules refers to a monolayer of amphiphilicmolecules having sufficient two-dimensional packing density so as tocreate a hydrophobic barrier between a surface to which the monolayer isbound and a hydrophilic liquid. As persons skilled in the art willappreciate, the appropriate packing density of a densely packedmonolayer will depend on the amphipathic molecules used. A denselypacked monolayer comprising alkyl-terminated siloxane, for example, willtypically comprise at least 1×10¹⁴ molecules/cm² (e.g., at least1.5×10¹⁴, 2.0×10¹⁴, 2.5×10¹⁴, or more molecules/cm²).

The amphiphilic molecules of a hydrophobic layer 510 can compriselong-chain hydrocarbons, which can be unbranched. Thus, the amphiphilicmolecules can comprise alkyl-terminated siloxane, alkyl-terminationphosphonic acid, or alkyl-terminated thiol. The long-chain hydrocarbonscan comprise a chain of at least 10 carbons (e.g., at least 16, 18, 20,22, or more carbons). In addition, the amphiphilic molecules cancomprise fluorinated (or perfluorinated) carbon chains. Thus, forexample, the amphiphilic molecules can comprise fluoroalkyl-terminatedsiloxane, fluoroalkyl-terminated phosphonic acid, orfluoroalkyl-terminated thiol. The fluorinated carbon chains can have thechemical formula CF₃—(CF₂)_(m)—(CH₂)_(n)—, wherein m is at least 2, n is0, 1, 2, or greater, and m+n is at least 9. For example, the fluorinatedcarbon chains can have the chemical formula CF₃—(CF₂)₇—(CH₂)₂—.

The monolayer of amphiphilic molecules, and thus the hydrophobic layer510 formed from such a monolayer, can have a thickness of less thanabout 5 nanometers (e.g., about 1.0 to about 4.0 nanometers, about 1.5to about 3.0 nanometers, or about 2.0 to about 2.5 nanometers).

Hydrophobic layers 510 formed from a monolayer of amphiphilic moleculescan be optionally patterned, such that select regions are relativelyhydrophilic compared to the remainder of the hydrophobic layer 510. Thiscan be achieved, for example, by applying a voltage potential across theSSOEW-configured substrate 500 for a period of time. Regions on thehydrophobic layer 510 that are illuminated and contacting an aqueousdroplet when the voltage potential is applied to the SSOEW-configuredsubstrate 500 will thereafter exhibit less hydrophobic (or relativelyhydrophilic) characteristics relative to the regions on the hydrophobiclayer 510 that were not contacting an aqueous droplet when the voltagepotential was applied. Without intending to be bound by theory, it isbelieved that the relatively hydrophilic regions comprise watermolecules that have intercalated into the amphiphilic monolayer. Aspersons skilled in the art will appreciate, the requisite voltagepotential for patterning the hydrophobic layer 510 will vary dependingupon the exact design of the SSOEW-configured substrate 500 (e.g., thethickness and impedances of the dielectric layer 506 and thephotoconductive layer 504, and the like). In certain embodiments, thevoltage potential required to pattern the hydrophobic layer 510 is atleast about 50 ppV (e.g., at least about 60 ppV, at least about 65 ppV,at least about 70 ppV, at least about 75 ppV, or at least about 80 ppV).

The photoconductive (or photosensitive) layer 504 (shown in FIGS. 3A-B,4A, and 5A-B) of an SSOEW-configured substrate 500 can comprise asemiconductor material that exhibits decreased electrical resistance inresponse to stimulation by electromagnetic radiation. Theelectromagnetic radiation can be, for example, radiation having awavelength in the visible spectrum and/or the near-infrared ornear-ultraviolet spectra. The semiconductor material can comprisesilicon. For example, the photoconductive layer 504 can comprisehydrogenated amorphous silicon (a-Si:H). The a-Si:H can comprise, forexample, about 8% to 40% hydrogen (i.e., calculated as 100*(the numberof hydrogen atoms)/(total number of hydrogen and silicon atoms)). Thea-Si:H photoconductive layer 504 can have a thickness of at least about500 nm (e.g., at least about 600 nm to about 1400 nm, about 700 nm toabout 1300 nm, about 800 nm to about 1200 nm, about 900 nm to about 1100nm, or about 1000 nm).

When the SSOEW-configured substrate 500 has a photoconductive layer 504formed from a layer of a-Si:H, the substrate 500 can optionally includefloating electrode pads located between the photoconductive layer 504and the dielectric layer 506. Such floating electrode pads have beendescribed, for example, in U.S. Pat. No. 6,958,132 (Chiou et al.).

Alternatively, the photoconductive layer 504 can comprise a plurality ofconductors, each conductor controllably connectable to the planarelectrode 502 of the SSOEW-configured substrate 500 via aphototransistor switch. Conductors controlled by phototransistorswitches are well-known in the art and have been described, e.g., inU.S. Patent Application No. 2014/0124370 (Short et al.), the contents ofwhich are incorporated herein by reference.

As will be appreciated by persons skilled in the art, the thickness ofthe photoconductive layer 504 can be varied in accordance with othercomponents of the SSOEW-configured substrate 500, such as the thicknessof the dielectric layer 506, so as to achieve suitable differencesbetween the impedance of the dielectric layer 506 and the impedance ofthe photoconductive layer 504 when the SSOEW-configured substrate 500 isin both the “on” or “illuminated” state (i.e., a voltage potential isapplied across the substrate 500 and the photoconductive layer 504 isilluminated) and the “off” or “dark” state (i.e., a voltage potential isapplied across the substrate 500 and the photoconductive layer 504 isnot illuminated). For example, the impedance of the dielectric layer 506can be tuned to have an impedance that is at least 10 times (e.g., atleast 15 times, at least 20 time, at least 25 times, at least 30 times,or more) greater than the impedance of the photoconductive layer 504 inthe illuminated state, and at least 10 times (e.g., (e.g., at least 15times, at least 20 time, at least 25 times, at least 30 times, or more)smaller than the impedance of the photoconductive layer 504 in the darkstate. As a particular example, the impedance of the dielectric layer506 can be about 10 kOhms to about 50 kOhms, and the impedance of thephotoconductive layer 504 (e.g., an a-Si:H photoconductive layer) can betuned to at least about 0.5 MOhms in the dark/off state and about 1kOhms or less in the illuminated/on state. These are only examples, butthey illustrate how the impedances can be tuned to achieve aSSOEW-configured substrate 500 displaying robust on/off performance.

The SSOEW-configured substrate 500 can comprise a planar electrode 502.The planar electrode 502 can comprising a conductive layer 502 a and,optionally, a support 502 b. For example, the conductive layer 502 a cancomprise (or consist essentially of, or consist of) a layer ofindium-tin-oxide (ITO). Alternatively, the conductive layer 502 a cancomprise a layer of electrically conductive silicon (e.g., highly p- orn-doped silicon). The support 502 b can be, for example, a layer ofglass or some other insulating material, such as a plastic. The planarelectrode 502 can comprise a single electrode (e.g., a single conductivelayer 502 a) or a plurality of individually addressable electrodes(e.g., two or more conductive layers 502 a that are spatially separatedfrom one another). The individually addressable electrodes can be, forexample, located on different regions of a common support 502 b, therebyproviding a SSOEW-configured substrate 500 having discreteSSOEW-configured regions. The individually addressable electrodes can beconnectable to one or more AC voltage sources via correspondingtransistor switches.

The planar electrode 502 (or a conductive layer 502 a thereof) and themesh electrode 508 of the SSOEW-configured substrate 500 can beconfigured to be connected to opposing terminals of an AC voltagesource, as shown in FIGS. 3B, 4A, and 5B. When the planar electrode 502(or a conductive layer 502 a thereof) and the mesh electrode 508 of theSSOEW-configured substrate 500 are connected to opposing terminals of anAC voltage source (shown in FIGS. 3B, 4A, and 5B), the substrate 500 iscapable of applying an electrowetting (EW) force to aqueous droplets incontact with the hydrophobic coating 510 of the substrate 500.Application of the force is controlled by illuminating specificlocations in the photoconductive layer 504 of the substrate 500. The ACvoltage used to achieve such EW-based movement of droplets will varydepending upon the exact construction of the substrate 500, and mayreflect the thickness and impedance of the dielectric layer 506 and thephotoconductive layer 504. In certain embodiments, a AC voltagepotential of at least 10 Volts peak-to-peak (ppV) (e.g., about 10 ppV toabout 80 ppV, about 20 ppV to about 70 ppV, about 25 ppV to about 60ppV, about 30 ppV to about 50 ppV, or about 40 ppV) is applied to thesubstrate in order to achieve droplet movement. The frequency of the ACvoltage potential also impacts the ability of the substrate 500 toachieve EW-based movement of droplets. Again, the frequency requirementscan vary depending upon the specific construction of the substrate 500.In certain embodiments, the AC voltage potential required to achievedroplet movement has a frequency of about 1 kHz to about 100 kHz (e.g.,about 2 kHz to about 80 kHz, about 3 kHz to about 60 kHz, about 4 kHz toabout 40 kHz, about 5 kHz to about 35 kHz, about 6 kHz to about 30 kHz,about 7 kHz to about 25 kHz, about 8 kHz to about 20 kHz, about 9 kHz toabout 15 kHz, or about 10 kHz). In certain embodiments, the voltagepotential applied to the substrate 500 to achieve droplet movement isabout 30 ppV to about 60 ppV (e.g., about 35 ppV to about 50 ppV, orabout 40 ppV) and has a frequency of about 5 kHz to about 35 kHz (e.g.,about 5 kHz to about 20 kHz, or about 10 kHz). By applying the foregoingAC voltage potentials to the SSOEW-configured substrates of theinvention, droplets can be moved about the surface of the hydrophobiccoating 510 at a rate of at least 0.01 mm/sec (e.g., at least 0.05mm/sec, at least 0.1 mm/sec, at least 0.5 mm/sec, at least 0.6 mm/sec,at least 0.7 mm/sec, at least 0.8 mm/sec, at least 0.9 mm/sec, at least1.0 mm/sec, at least 1.5 mm/sec, at least 2.0 mm/sec, at least 2.5mm/sec, at least 3.0 mm/sec, at least 3.5 mm/sec, at least 4.0 mm/sec,at least 4.5 mm/sec, at least 5.0 mm/sec, or more).

FIGS. 4A-B illustrate circuit models for a SSOEW-configured substrate500 of the invention. As shown, a conductive layer 502 a of the planarelectrode 502 is electrically connected to droplet 520, which is restingon the hydrophobic coating 510, via the photoconductive layer 504, thedielectric layer 506, and the hydrophobic coating 510. The resistance inthe photoconductive layer 504 is variable, depending on whether or notlight is incident on a particular region of the photoconductive layer504, and thus there is a voltage drop across the photoconductive layer504 which varies depending upon the resistance R of the photoconductivelayer 504. The dielectric layer 506 and the hydrophobic coating 510function as capacitors C1 and C2, respectively, in the electricalcircuit. In addition, there is capacitive connection C2′ between thedroplet 520 and the mesh electrode 510. The AC voltage source completesthe electrical circuit by connecting to both the mesh electrode 508 andthe conductive layer 502 a of the planar electrode 502. Voltage droppedacross the dielectric layer 506 results in droplet actuation. Voltagedropped across shunt connections (e.g., between the conductive layer 502a of the planar electrode 502 and the wires of the mesh electrode 508,through the dielectric layer 506 and the photoconductive layer 504) (notshown) are not useful for actuation. Accordingly, it is advantageous tominimize the area of the dielectric layer 506 that is shielded from thedroplet 520 by the wires of the mesh electrode 508. Assuming a linearfill factor β for the mesh electrode 508, the electrowetting force isapproximately (1−β) times the electrowetting force of an OEW devicehaving a top electrode. Thus, for example, for a mesh electrode 508 madefrom 10 micron-wide wires on a 1 mm pitch yields a linear fill factorβ=99%. Accordingly, by keeping the wires of the mesh electrode 508 thinand spread out, the electrowetting force of the SSOEW-configuredsubstrate 500 is substantially unchanged relative to an OEW devicehaving a planar top electrode.

The pitch of the wires in the mesh electrode 508 also impacts the sizeof the droplets that can be moved on the SSOEW-configured substrate 500.In general, the pitch of the wires should be smaller than the diameterof the droplet, otherwise the droplet can get trapped between wires.FIG. 9 shows the relationship between the minimum droplet volume and thepitch of the mesh electrode 508. As can be seen, the movement ofnanoliter-scale droplets (e.g., 100 nL up to 500 nL/droplet) willtypically require the wires of the mesh electrode 508 to have a pitch ofabout 100 microns to about 500 microns. Likewise, the movement ofpicoliter-scale droplets (e.g., 100 pL to 500 pL/droplet) will typicallyrequire the wires of the mesh electrode 508 to have a pitch of about 5microns to about 50 microns. The relationship between the pitch of thewires and the maximum wire width that allows for a linear fill factor βof at least 90% is also shown in FIG. 9.

Microfluidic Devices Having a SSOEW-Configured Substrate Base.

The SSOEW-configured substrates of the invention can be integrated intomicrofluidic devices. For example, the SSOEW-configured substrate canprovide a base for the microfluidic device. The microfluidic device canfurther include walls, disposed upon the substrate/base, that extendvertically upward from the substrate/base. Together, the walls and thesubstrate/base can define a microfluidic circuit configured to hold aliquid medium. The liquid medium can be, for example, a hydrophobicliquid, such as an oil. In addition, the microfluidic circuit can hold adroplet of liquid, such as an aqueous medium. Typically, the liquidmedium and the liquid of the droplet are selected to be immiscibleliquids.

The walls of the microfluidic device can define one or more flowregions, which can be microfluidic channels. In addition, the walls canfurther define one or more (e.g., a plurality of) chambers in themicrofluidic device, each fluidically connected to and opening off of atleast one flow region (or microfluidic channel). One or more suchchambers can be a sequestration pen. Thus, for example, the walls candefine a single microfluidic channel and a plurality of chambersfluidically connected thereto, or a plurality of microfluidic channelswith each channel fluidically connected to a plurality of chambers.Furthermore, each chamber can be fluidically connected to more than onemicrofluidic channel, as illustrated in FIGS. 7 and 8.

The walls of the microfluidic device can comprise a structural polymer.As used herein, the term “structural polymer” refers to materials thatcomprise a polymer and have sufficient structural rigidity so as to formstructures that have a height that is greater than a width of thestructure. For example, a structural polymer can form a wall which, whenviewed in cross-section, has an aspect ratio of about 1:1.1, about1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:2, about 1:2.5,about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, or greater.The structural polymer can comprise (or consist essentially of, orconsist of), for example, a silicon-based polymer, such aspolydimethylsiloxane (PDMS) or photo-patternable silicone (PPS), bothavailable from Dow Corning. Alternatively, the walls can comprise anepoxy-based adhesive. The epoxy-based adhesive can be, for example, SU-8or equivalent types of adhesives.

The walls of the microfluidic device can have a height of at least about30 microns, at least about 40 microns, at least about 50 microns, atleast about 60 microns, at least about 70 microns, at least about 80microns, at least about 90 microns, at least about 100 microns, or more.Thus, for example, the height of the walls can be about 30 to about 60microns, about 40 to about 80 microns, about 50 to about 100 microns,about 60 to about 120 microns, about 70 to about 140 microns, about 75to about 150 microns, about 80 to about 160 microns, about 90 into about180 microns, or about 100 to about 200 microns. In addition, the wallscan have a cross-sectional width of about 10 to about 50 microns, orabout 20 to about 40 microns.

The microfluidic device can optionally include a cover, which can bedisposed on the walls. The cover can be substantially parallel to thesubstrate/base. Together, the substrate, the walls, and the cover candefine an enclosure configured to contain a liquid medium. The liquidmedium can be, for example, a hydrophobic liquid, such as an oil. Inaddition, the enclosure can contain a droplet of liquid, such as anaqueous medium. Typically, the liquid medium and the liquid of thedroplet are selected to be immiscible liquids.

The cover of the microfluidic device can be made, all or in part, fromthe same material(s) as the walls. Thus, the cover can comprise astructural polymer, such as a silicon-based polymer (e.g., PDMS or PPS).Alternatively, the cover can be made, all or in part, from material(s)that differ from the wall material(s), such as rigid material (e.g.,glass). The cover can be made, at least in part, from material that ispiercable, so that droplets can be introduced or removed from enclosureby a piercing structure, such as a needle. The cover can comprise ahydrophobic coating, such as the hydrophobic coating 510 of theSSOEW-configured substrate 500.

In some embodiments, the cover can be a SSOEW-configured substrate 500.Thus, the microfluidic device can have both the base and the coverconfigured to provide an electrowetting force to an aqueous dropletlocated within the enclosure.

A microfluidic device of the invention can be manufactured, for example,by: bonding wall material to the dielectric layer 506 (and, if exposed,the mesh electrode 508) of a SSOEW-configured substrate 500 that islacking the hydrophobic coating 510; optionally bonding the wallmaterial to a cover; and applying the hydrophobic coating 510 on theportions of the dielectric layer 506 (and mesh electrode 508) thatremain exposed after bonding of the wall material. For the followingdiscussion regarding the application of the hydrophobic coating 510 tothe dielectric layer 506 (and, if exposed, the mesh electrode 508), itshould be understood that the referenced SSOEW-configured substrate 500(or simply substrate 500) may not include the hydrophobic coating 510,but such should be evident from the nature of the discussion.

The wall material can be applied directly to the dielectric layer 506(and, if exposed, the mesh electrode 508), or it can be applied to thecover (if present) and then subsequently applied to the dielectric layer506 (and mesh electrode 508). The wall material can be applied initiallyas a continuous layer and then patterned (e.g., by photo-patterning oretching, depending upon the type of wall material used).

The hydrophobic coating 510 can be applied via dip/spin coating, spraycoating, vapor deposition, or the like. For example, a hydrophobiccoating 510 comprising polytetrafluoro-ethylene (PTFE) (i.e., Teflon®)or poly(2,3-difluoromethylenyl-perfluorotetrahydrofuran) (i.e., Cytop™)can be applied by spin coating. Deposition of amphiphilic molecules,such as alkyl- or fluoroalkyl-terminated siloxanes or alkyl- orfluoroalkyl-terminated thiols can be achieved by vapor deposition.

Application of the hydrophobic coating 510 by dip/spin coating generallycomprises dipping the dielectric layer 506 of the SSOEW-configuredsubstrate 500 into a solution comprising molecules of the hydrophobiccoating diluted in a solvent (e.g., polytetrafluoro-ethylene (PTFE) orpoly(2,3-difluoromethylenyl-perfluorotetrahydrofuran)), spinning thecoated substrate 500 in a centrifuge, and then baking the substrate at atemperature high enough and for a duration long enough to boil off thesolvent. The exact conditions will vary with the coating being applied.With Teflon® AF (Dupont), the coating is provided at a concentration ofabout 6% and is diluted in a fluorinated oil, such as Fluorinert-40(FC-40) or Fluorinert-77 (FC-77), to a final concentration of less than0.5% (e.g., about 0.2%). With Cytop™ (CTL-09M, Dupont), the coating isprovided at a concentration of about 9% and is diluted in CTSOLV-180 orCTSOLV-100E BP Perfluorinated Solvent to a final concentration of about2% or less (e.g., about 0.1% to about 2.0%). Of course, theseCytop™-specific solvents could be replaced by conventional fluorinatedoils, such as FC-40 or FC-77. After the dielectric layer 506 of thesubstrate 500 is dipped in the diluted coating solution, the substratecan be centrifuged at a speed (e.g., about 3000 RPM) and for a durationof time (e.g., at least 15 seconds, about 20 seconds to about 1 minute,or about 30 seconds) sufficient to produce a substantially uniformcoating on the dielectric layer 506. Excess solvent is then baked off ata temperature appropriate for the solvent used and for a durationsufficient to achieve the desired coating (e.g., at least 20 minutes,about 20 minutes to about 1 hour, or about 30 minutes). The bakingtemperature for CTSOLV-180 and CTSOLV-100E, for example, are about 180°C. and 100° C., respectively.

Application of a monolayer of amphiphilic molecules can be performed byvapor deposition, with the exact procedure varying according to theamphiphilic molecules being used. For example, for alkyl- orfluoroalkyl-terminated siloxanes, the deposition can be performed at atemperature of at least about 110° C. (e.g., at least about 120° C., atleast about 130° C., at least about 140° C., at least about 150° C., atleast about 160° C., etc.), for a period of at least about 15 hours(e.g., at least about 20 hours, at least about 25 hours, at least about30 hours, at least about 35 hours, at least about 40 hours, or at leastabout 45 hours). Such vapor deposition is typically performed undervacuum and in the presence of a water source, such as a hydrated salt(e.g., MgSO₄.7H₂O). Typically, increasing the temperature and durationof the vapor deposition produces improved characteristics of thehydrophobic layers 122 and 142, but such parameters can be significantlyimpacted by the vacuum chamber used. The vapor deposition process can beoptionally improved by pre-cleaning the dielectric layer. Suchpre-cleaning can include a solvent bath, such as an acetone bath, anethanol bath, or both (e.g., sequentially). The pre-cleaning can includesonication during the solvent bath. Alternatively, or in addition, suchpre-cleaning can include treating the substrate 500 in an oxygen plasmacleaner. The oxygen plasma cleaner can be operated under vacuumconditions, at a power setting (e.g., 100 W) and for a duration (e.g.,at least 30 seconds, at least 45 seconds, at least 60 seconds, or more)sufficient to produce a clean surface on the dielectric layer 506.

Application of a monolayer of amphiphilic molecules can be performed bydip coating as well. For example, alkyl- or fluoroalkyl-terminatedphosphonic acids or alkyl- or fluoroalkyl-terminated thiols can beapplied by dip coating. The dip coating process can be optionallyimproved by pre-cleaning the dielectric layer 506 of theSSOEW-configured substrate 500. Such pre-cleaning can include a solventbath, such as an acetone bath, an ethanol bath, or both (e.g.,sequentially). The pre-cleaning can include sonication during thesolvent bath. Alternatively, or in addition, such pre-cleaning caninclude treating the substrate 500 in an oxygen plasma cleaner. Theoxygen plasma cleaner can be operated under vacuum conditions, at apower setting (e.g., 100 W) and for a duration (e.g., at least 30second, at least 45 seconds, at least 60 seconds, or more) sufficient toproduce a clean surface on the dielectric layer 506. The substrate 500is then submerged in a freshly prepared 10 mM solution of theamphiphilic molecule in ethanol for a period of time sufficient toachieve formation of the desired densely-packed monolayer. Afterremoving the substrate 500 from the solution, the surfaces of thesubstrate 500 are rinsed with an excess amount of ethanol.

Methods for Moving Droplets in a Microfluidic Device.

FIG. 7 illustrates an example of a microfluidic device 700 whichcomprises: a microfluidic circuit having microfluidic channels 712, 714and a plurality of chambers 716; and a droplet generator 706 forproviding fluidic droplets 720 to the microfluidic circuit. Microfluidicchannel 714 is configured to hold a first fluidic medium 724. Typically,the first fluidic medium is a hydrophobic fluid, such as an oil (e.g., asilicone oil or a fluorinated oil). Microfluidic channel 714 isconnected to the droplet generator 706 via an interface 708, whichallows channel 714 to receive droplets 720 generated by the dropletgenerator 706. The received droplets 720 comprise a liquid which isimmiscible in the first fluidic medium 724. Typically, the receiveddroplets will comprise an aqueous medium, which may containmicro-objects, such as cells or beads, or reagents that are soluble inaqueous media. Microfluidic channel 714 is also connected to each of theplurality of chambers 716, facilitating movement of received droplets720 (as well as droplets 732 pulled from a reservoir of fluid immisciblein the first fluidic medium 724) into and between chambers 716.

Microfluidic channel 712 of device 700 is connected to a subset ofchambers 716, and thus is indirectly connected to microfluidic channel714 via such chambers 716. As illustrated, microfluidic channel 712 andthe chambers 716 connected thereto contains a fluidic medium 722 whichis immiscible in the first fluidic medium 724. Thus, for example,fluidic medium 722 can be an aqueous medium, such as a cell culturemedium. When fluidic medium 722 is a cell culture medium, the chambers716 that contain culture medium can be used as culture chambers forgrowing cells, and microfluidic channel 712 can be a perfusion channelthat provides a flow of fresh culture medium. As discussed herein, theflow of fresh culture medium in a perfusion channel can, via diffusionof molecules between the perfusion channel and a culture chamber,provide nutrients to the chamber and remove waste from the chamber, thusfacilitating continued cell growth.

FIG. 8 illustrates another example of a microfluidic device 800 of theinvention. Device 800 comprises: a microfluidic circuit havingmicrofluidic channels 712, 714, a first plurality of chambers 816, and asecond plurality of chambers 716; and a droplet generator 706 forproviding fluidic droplets 720 to the enclosure. FIG. 8 presents avariation on the microfluidic apparatus 700 shown in FIG. 7, whereinchambers 716 contain a medium 722 that is immiscible in the firstfluidic medium 724 (located in microfluidic channel 714) and are locateddirectly across the microfluidic channel 714 from corresponding chambers814. This configuration facilitates movement of fluid droplets 732(optionally containing micro-objects 730 or biological material) from aselect chamber 716 to the corresponding chamber 816, where the fluiddroplets (and any micro-objects 730 or biological material) can beprocessed.

The microfluidic circuits formed by the microfluidic channels 712, 714and chambers 716, 816 are merely examples, and many other configurationsof channels and chambers are encompassed by the invention. For example,in each of apparatuses 700 and 800, microfluidic channel 712 and thechambers 716 directly connected to channel 712 are optional features.Thus, apparatuses 700 and 800 can lack perfusion channels and culturechambers.

In embodiments where microfluidic channel 712 is present, the substratewhich helps to define channel 712 and/or directly connected chambers 716(e.g., by forming the base of the channel and/or chambers) can have anelectrowetting configuration. Alternatively, however, the substratewhich helps to define the channel 712 and/or directly connected chambers716 can lack an electrowetting configuration (e.g., and instead can havea DEP configuration, or neither an electrowetting nor a DEPconfiguration). In embodiments in which microfluidic channel 712 ispresent, and the substrate which helps to define channel 712 and/ordirectly connected chambers 716 has an electrowetting configuration, thehydrophobic coating 510 of the substrate can be patterned to be morehydrophilic than the hydrophobic coating of the substrate which helps todefine channel 714. The increased hydrophilicity can be achieved, forexample, as discussed above.

The droplet generator 706 and any microfluidic circuit to which itprovides droplets can be part of a microfluidic device (either anintegral part or connected thereto), which can be like any of themicrofluidic devices illustrated in the drawings or described herein.Although one droplet generator 706 is shown in FIGS. 7 and 8, more thanone such droplet generator 706 can provide droplets to the microfluidiccircuit of apparatuses 700 and 800. The droplet generator 706 itself caninclude an electrowetting configuration, and can thus comprise asubstrate having a photoresponsive layer, which can comprise a-Si:H(e.g., as illustrated in U.S. Pat. No. 6,958,132), a photo-actuatedcircuit substrate (e.g., as illustrated in U.S. Patent ApplicationPublication No. 2014/0124370), a phototransistor-based substrate (e.g.,as illustrated in U.S. Pat. No. 7,956,339), or an electrically-actuatedcircuit substrate (e.g., as illustrated in U.S. Pat. No. 8,685,344).Alternatively, the droplet generator can have a T- or Y-shapedhydrodynamic structure (e.g., as illustrated in U.S. Pat. Nos.7,708,949, 7,041,481 (reissued as RE41,780), 2008/0014589, 2008/0003142,2010/0137163, and 2010/0172803). All of the foregoing U.S. patentdocuments are incorporated by reference herein in their entirety.

As shown, the droplet generator 706 can comprise one or more fluidicinputs 702 and 704 (two are shown but there can be fewer or more) and afluidic output 708, which can be connected to the microfluidic channel714. Liquid media 722, 724, biological micro-objects 730, reagents,and/or other biological media can be loaded through the inputs 702 and704 into the droplet generator 706. The droplet generator 706 cangenerate and output into the channel 714 droplets 720 of the liquidmedium 722 (which can, but need not, contain one or more biologicalmicro-objects 730), reagents, or other biological medium. If the channel714 has an electrowetting configuration, the droplets 720 can be movedin the channel 714 utilizing electrowetting (or optoelectrowetting).Alternatively, the droplets 720 can be moved in the channel 714 by othermeans. For example, the droplets 720 can be moved in the channel 714using fluidic flow, gravity (if the microfluidic device includes acover), or the like.

As discussed above, the microfluidic channel 714 and select chambers716/816 can be filled with a first fluidic medium 724, and microfluidicchannel 712 and chambers 716 connected directly thereto can be filledwith a second fluidic medium 722. The second fluidic medium 722(hereinafter an “aqueous medium”) can be an aqueous medium, such as asample medium for maintaining, culturing, or the like biologicalmicro-objects 730. The first fluidic medium 724 (hereinafter an“immiscible medium”) can be a medium in which the aqueous medium 722 isimmiscible. Examples of the aqueous medium 722 and the immiscible medium724 include any of the examples discussed above for various media.

The droplet generator 706 can be utilized to load biologicalmicro-objects and/or facilitate the running of biochemical and/ormolecular biological workflows on the microfluidic apparatus. FIGS. 7and 8 illustrate non-limiting examples. By using a droplet generator,the apparatus can have an electrowetting configuration throughout thefluidic circuit.

FIGS. 7 and 8 illustrate an example in which the droplet generator 706generates a droplet 720 comprising a reagent (or other biologicalmaterial). The reagent-containing droplet 720 can be moved through themicrofluidic channel 714 and into one of the chambers 716/816 containingthe immiscible medium 724. Prior to or after moving thereagent-containing droplet 720 into one of the chambers 716/816, one ormore micro-objects 730 in one or more droplets 732 can be moved into thesame chambers 716/816. The reagent-containing droplet 720 can then bemerged with the droplet 732 containing the micro-object 730, allowingthe reagents of droplet 720 to mix and chemically react with thecontents of droplet 732. The one or more micro-object-containingdroplets 732 can be supplied by the droplet generator 706 or can beobtained from a holding pen 716, as shown in FIGS. 7 and 8. Themicro-object 730 can be a biological micro-object, such as a cell, whichhas optionally been cultured (e.g., in a chamber 716) prior to beingmoved to the processing chamber 716/816. Alternatively, the micro-object730 can be a bead, such as an affinity bead that is capable of bindingto molecules of interest in a sample (e.g., cell secretions present inculture medium 722 after the culture medium 722 has been used to cultureone or more biological cells). In still other alternatives, the one ormore droplets 732 can contain no micro-objects but only aqueous medium,such as culture medium 722, e.g., that contains cell secretions afterthe culture medium 722 has been used to culture one or more biologicalcells.

Various processes can be performed in a microfluidic device comprising amicrofluidic circuit like any of devices 700 and 800.

At a step 402 of a process 400, a biological micro-object can becultured in a holding pen filled with a sample medium (e.g., cellculture medium). For example, a micro-object 730 of FIG. 7 or 8 can be abiological cell and can be cultured in its chamber 716. Culturing can begenerally as discussed above. For example, culturing can includeperfusing the channel 712 with a culture medium 722. Step 402 can beperformed over a specified period of time.

At a step 404, the cultured biological micro-object can be moved fromthe sample-medium-filled chamber 716 in which it was cultured to achamber 716/816 filled with a medium in which the sample medium isimmiscible. For example, the cultured micro-object 730 can be moved in adroplet 720 or 732 of culture medium 722 from one of the holding pens716 into one of the holding pens 716/816, as illustrated in FIGS. 7 and8, as discussed above.

At a step 406, the cultured biological micro-object can be subjected toone or more treatments or processes in the immiscible-medium-filledholding pen. For example, one or more droplets 720 containing one ormore reagents can be produced by the droplet generator 706 and movedinto an immiscible-medium-filled chamber 712/816 and merged with thedroplet 732 containing the cultured biological micro-object 730, asshown in FIGS. 7 and 8 and discussed above. For example, a firstreagent-containing droplet 720 can contain a lysing reagent. Merger ofthe droplet 732 containing the cultured biological micro-object 730 withthe first reagent-containing droplet 720 containing lysing reagent,would result in the lysis of the cultured biological micro-object 730.In other words, a combined droplet (not shown) would be formed thatcontains a cell lysate from the cultured biological micro-object 730.Additional (e.g., second, third, fourth, etc.) reagent-containingdroplets 720 could then be merged with the cell lysate-containing newdroplet, so as to further process the cell lysate as desired.

In addition or as another example, one or more droplets containing oneor more labeled capture micro-objects (not shown) having an affinity fora secretion or other material or materials of interest (e.g., nucleicacids such as DNA or RNA, proteins, metabolites, or other biologicalmolecules) produced the cultured biological micro-object 730 can begenerated by the droplet generator 706 and moved into theimmiscible-medium-filled pen 716 or 816 and merged with the droplet ofculture medium 722 containing the cultured biological micro-object 730in a similar manner. In cases where the cultured biological micro-object730 has already been lysed, capture micro-object-containing droplet 720could contain one or more affinity beads (e.g., having affinity fornucleic acids, such as DNA, RNA, microRNAs, or the like) which, uponmerger with the cell lysate-containing droplet in holding pen 716 or816, could bind to target molecules present in the lysate.

At a step 408, the treated biological micro-object can be optionallyprocessed. For example, if at step 406, a capture object (not shown) ismoved into the immiscible-medium-filled chamber 716/816 with thecultured biological micro-object 730, the chamber 716/816 can bemonitored at step 408 for a reaction (e.g., a fluorescent signal)indicative of a quantity of the material of interest bound to thelabeled capture micro-object. Alternatively, such a capture micro-object(not shown) can be removed (e.g., in a droplet 722) from the chamber716/816 and exported from the microfluidic device (not shown in FIG. 7or 8) for subsequent analysis. As yet another example, the treatedbiological micro-object 730 can be removed (e.g., in a droplet 732) fromthe chamber 716/816 and exported from the microfluidic device (notshown) for subsequent analysis.

Although specific embodiments and applications of the invention havebeen described in this specification, these embodiments and applicationsare exemplary only, and many variations are possible. For example, themethods of FIG. 4 can be performed with respect to culture medium thatcontains cell secretions (e.g., after the culture medium 722 has beenused to culture one or more biological cells). In such an embodiment,step 402 would remain the same, but step 404 would involve movingdroplets 732 which can contain no micro-objects but only aqueous medium,such as culture medium 722 containing cell secretions, intoimmiscible-medium-containing chambers 716/816, and steps 406 and 408would be performed with respect to such aqueous medium-containingdroplets 732.

EXAMPLES Example 1 Movement of Water Droplets on a SSOEW-ConfiguredSubstrate

The movement of a 1 microliter droplet of water (conductivity 10 mS/m)was successfully demonstrated on the device of FIG. 1. The devicefeatured a gold mesh ground that had a thickness of substantially 50 nm.Individual wires of the mesh had a width of substantially 10 microns andpitch of substantially 2 mm (β=0.5%). Prior to operation, the surface ofthe device was primed with silicone oil to reduce friction.

An AC voltage bias was applied between the ITO electrode and the meshground electrode. Structured light was then projected onto the substrateproximal to the droplet being moved, such that the light at leastpartially contacted the droplet. Droplets were successfully moved byshifting the location of the structured light. In this experiment, lighthaving a square shape roughly the same size as the droplet was used. Amaximum velocity of 0.33 cm/s was measured using a 40 ppV bias at 10kHz.

The experiment confirmed that the droplets were free to move in anyarbitrary direction so long as the droplet maintained electrical contactwith the mesh ground electrode. As shown in FIGS. 10A-B, droplets weremoved along rectangular and circular paths. In addition, it was possibleto merge droplets using the device.

Example 2 Movement of PBS Droplets on a SSOEW-Configured Substrate

Subsequently, a SSOEW-configured substrate was built having aphotoconductive layer of substantially 1 micron thickness, a firstdielectric layer of substantially 150 nm thickness, a second dielectriclayer of substantially 2.5 nm in thickness, a mesh electrode havingwires consisting of a gold layer of substantially 25 nm in thicknesswith an underlying titanium layer of substantially 10 nm in thickness,and a hydrophobic coating comprising a monolayer ofoctadecyltrimethoxysilane. The photoconductive layer consistedessentially of hydrogenated amorphous silicon (a-Si:H), and the firstand second dielectric layers were made from Alumina (i.e., aluminumoxide deposited by ALD). The mesh electrode was interposed between thefirst and second dielectric layers, and the wires of the mesh electrodewere substantially 10 microns in width, with a pitch of approximately300 microns.

A droplet of PBS (approximately 1.5 microliters in volume) wassuccessfully moved around the surface of the SSOEW-configured substrateat a rate of about 1 mm/s. To achieve this movement, an AC voltagepotential of about 40 ppV, with frequency of about 10 kHz, was appliedto the substrate. The PBS droplet was moved through 3 cSt silicone oil(with the substrate submerged therein).

From the description herein, it will be appreciated that that thepresent disclosure encompasses multiple embodiments which include, butare not limited to, the following:

1. A substrate comprising: a planar electrode; a photoconductive layer;a dielectric layer; a mesh ground electrode; and a hydrophobic coating;wherein the photoconductive layer is interposed between the planarelectrode and the dielectric layer, with a bottom surface of thephotoconductive layer adjoining a top surface of the planar electrodeand a top surface of the photoconductive layer adjoining a bottomsurface of the dielectric layer; wherein the mesh ground electrodeadjoins a top surface of the dielectric layer; wherein the planarelectrode and the mesh ground electrode are configured to be connectedto an AC voltage source; and

wherein, when the planar electrode and the mesh ground electrode areconnected to opposing terminals of the AC voltage source, the substrateis capable of applying an opto-electrowetting (OEW) force to aqueousdroplets in contact with the hydrophobic coating.

2. The substrate of any preceding embodiment, wherein the substrateforms all or part of the base of a microfluidic device.

3. The substrate of any preceding embodiment, wherein the planarelectrode comprises a metal conductor.

4. The substrate of any preceding embodiment, wherein the planarelectrode comprises an indium-tin-oxide (ITO) layer.

5. The substrate of any preceding embodiment, wherein the planarelectrode comprises a non-metal conductor.

6. The substrate of any preceding embodiment, wherein the planarelectrode comprises a layer of conductive silicon.

7. The substrate of any preceding embodiment, wherein thephotoconductive layer comprises hydrogenated amorphous silicon (a-Si:H).

8. The substrate of any preceding embodiment, wherein thephotoconductive layer has a thickness of at least 500 nm.

9. The substrate of any preceding embodiment, wherein thephotoconductive layer has a thickness of about 900 to 1100 nanometers.

10. The substrate of any preceding embodiment, wherein the dielectriclayer comprises a metal oxide.

11. The substrate of any preceding embodiment, wherein the dielectriclayer comprises aluminum oxide.

12. The substrate of any preceding embodiment, wherein the dielectriclayer has a thickness of at least 125 nm.

13. The substrate of any preceding embodiment, wherein the dielectriclayer was formed by atomic layer deposition.

14. The substrate of any preceding embodiment, wherein the dielectriclayer is a composite dielectric layer having at least a first dielectriclayer and a second dielectric layer, with a bottom surface of the firstdielectric layer adjoining the photoconductive layer.

15. The substrate of any preceding embodiment, wherein the mesh groundelectrode is interposed between the first dielectric layer and thesecond dielectric layer.

16. The substrate of any preceding embodiment, wherein the compositedielectric layer has a thickness of at least 125 nm.

17. The substrate of any preceding embodiment, wherein the first andsecond dielectric layers both comprise a metal oxide.

18. The substrate of any preceding embodiment, wherein the first andsecond dielectric layers both comprise aluminum oxide.

19. The substrate of any preceding embodiment, wherein the first andsecond dielectric layers are both formed by atomic layer deposition.

20. The substrate of any preceding embodiment, wherein the firstdielectric layer has a thickness of about 125 nm to about 175 nm

21. The substrate of any preceding embodiment, wherein the seconddielectric layer has a thickness of less than 10 nm.

22. The substrate of any preceding embodiment, wherein the firstdielectric layer has a top surface adjoining a bottom surface of themesh ground electrode.

23. The substrate of any preceding embodiment, wherein the firstdielectric material forms a lattice.

24. The substrate of any preceding embodiment, wherein the top surfaceof the first dielectric layer is substantially contiguous with thebottom surface of the mesh ground electrode.

25. The substrate of any preceding embodiment, wherein the firstdielectric layer is made from a material having a dielectric constant∈1, the second dielectric layer is made from a material having adielectric constant ∈2, and ∈1 is less than ∈2.

26. The substrate of any preceding embodiment, wherein the firstdielectric layer comprises a metal oxide.

27. The substrate of any preceding embodiment, wherein the firstdielectric layer comprises aluminum oxide.

28. The substrate of any preceding embodiment, wherein the firstdielectric layer has a thickness of about 50 nm to about 150 nm.

29. The substrate of any preceding embodiment, wherein the seconddielectric layer comprises a non-metal oxide.

30. The substrate of any preceding embodiment, wherein the seconddielectric layer comprises silicon oxide.

31. The substrate of any preceding embodiment, wherein the compositedielectric layer comprises a first dielectric layer, a second dielectriclayer, and a third dielectric layer, with the second dielectric layerinterposed between the first and third dielectric layers.

32. The substrate of any preceding embodiment, wherein the first andthird dielectric layers each comprise a metal oxide.

33. The substrate of any preceding embodiment, wherein the first andthird dielectric layers each comprise aluminum oxide.

34. The substrate of any preceding embodiment, wherein the first andthird dielectric layers are each formed by atomic layer deposition.

35. The substrate of any one of any preceding embodiment, wherein thefirst and third dielectric layers each have a thickness of at least 10nanometers.

36. The substrate of any preceding embodiment, wherein the first andthird dielectric layers each have a thickness of about 10 to 20nanometers.

37. The substrate of any one of any preceding embodiment, wherein thesecond dielectric layer comprises a non-metal oxide or a nitride.

38. The substrate of any preceding embodiment, wherein the seconddielectric layer comprises silicon oxide.

39. The substrate of any preceding embodiment, wherein the seconddielectric layer is formed by plasma enhanced chemical vapor deposition.

40. The substrate of any preceding embodiment, wherein the seconddielectric layer has a thickness of at least 100 nanometers.

41. The substrate of any preceding embodiment, wherein the firstdielectric layer has a top surface adjoining a bottom surface of themesh ground electrode, and the third dielectric layer has a bottomsurface adjoining a top surface of the mesh ground electrode.

42. The substrate of any preceding embodiment, wherein the firstdielectric layer has a dielectric constant ∈1, the third dielectriclayer has a dielectric constant ∈3, and ∈1 is less than ∈3.

43. The substrate of any preceding embodiment 42, wherein the seconddielectric layer has a dielectric constant ∈2, and ∈2 is less than ∈3.

44. The substrate of any preceding embodiment, wherein the mesh groundelectrode comprises wires that are arranged in a lattice shape.

45. The substrate of any preceding embodiment, wherein the mesh groundelectrode further comprises plates located on top of vertices formed bythe wires of the mesh ground electrode.

46. The substrate of any preceding embodiment, wherein the wires of themesh ground electrode have a substantially square shape or asubstantially rectangular shape in cross-section.

47. The substrate of any preceding embodiment, wherein the wires of themesh ground electrode, in cross section, have an average width and anaverage height, with the average height being at least 50 nm.

48. The substrate of any preceding embodiment, wherein the wires of themesh ground electrode have a T-shape in cross section.

49. The substrate of any preceding embodiment, wherein the mesh groundelectrode comprises a conductive metal.

50. The substrate of any preceding embodiment, wherein the mesh groundelectrode comprises gold or aluminum.

51. The substrate of any preceding embodiment, wherein the mesh groundelectrode comprises aluminum that has an oxidized outer surface.

52. The substrate of any preceding embodiment, wherein the mesh groundelectrode has a linear fill factor β less than or equal to 10%.

53. The substrate of any preceding embodiment, wherein wires of the meshground electrode have a pitch of about 200 microns to about 500 microns.

54. The substrate of any preceding embodiment, wherein the hydrophobiccoating has a bottom surface that adjoins at least a portion of a topsurface of the dielectric layer.

55. The substrate of any preceding embodiment, wherein the bottomsurface of the hydrophobic coating adjoins a top surface of the meshground electrode.

56. The substrate of any preceding embodiment, wherein the hydrophobiccoating comprises an organofluorine polymer having at least oneperfluorinated segment.

57. The substrate of any preceding embodiment, wherein theorganofluorine polymer comprises polytetrafluoro-ethylene (PTFE) orpoly(2,3-difluoromethylenyl-perfluorotetrahydrofuran).

58. The substrate of any preceding embodiment, wherein the hydrophobiccoating has a thickness of at least 20 nm.

59. The substrate of any preceding embodiment, wherein the hydrophobiclayer comprises a densely packed monolayer of amphiphilic moleculescovalently bonded to molecules of the dielectric layer.

60. The substrate of any preceding embodiment, wherein the amphiphilicmolecules of the hydrophobic layer each comprise a siloxane group, andwherein the siloxane groups are covalently bonded to the molecules ofthe dielectric layer.

61. The substrate of any preceding embodiment, wherein the amphiphilicmolecules of the hydrophobic layer each comprise a phosphonic acidgroup, and wherein the phosphonic acid groups are covalently bonded tothe molecules of the dielectric layer.

62. The substrate of any preceding embodiment, wherein the amphiphilicmolecules of the hydrophobic layer each comprise a thiol group, andwherein the thiol groups are covalently bonded to the molecules of thedielectric layer and/or the mesh ground electrode.

63. The substrate of any preceding embodiment, wherein the amphiphilicmolecules of the hydrophobic layer comprise long-chain hydrocarbons.

64. The substrate of any preceding embodiment, wherein the long chainhydrocarbons are unbranched.

65. The substrate of any preceding embodiment, wherein the long-chainhydrocarbons comprise a chain of at least 10 carbons.

66. The substrate of any preceding embodiment, wherein the long-chainhydrocarbons comprise a chain of at least 16 carbons.

67. The substrate of any preceding embodiment, wherein the long-chainhydrocarbons comprise a chain of 16, 18, 20, or 22 carbons.

68. The substrate of any preceding embodiment, wherein the amphiphilicmolecules of the hydrophobic layer comprise fluorinated carbon chains.

69. The substrate of any preceding embodiment, wherein the fluorinatedcarbon chains have the chemical formula CF3-(CF2)m-(CH2)n-, wherein m isat least 2, n is at least 2, and m+n is at least 9.

70. The substrate of any preceding embodiment, wherein the fluorinatedcarbon chains have the chemical formula CF3-(CF2)m-(CH2)n-, wherein m isat least 7 and n is at least 2.

71. The substrate of any preceding embodiment, wherein the hydrophobiclayer has a thickness of less than 5 nanometers.

72. The substrate of any preceding embodiment, wherein the hydrophobiclayer is patterned such that select regions are relatively hydrophiliccompared to the remainder of the hydrophobic layer.

73. A microfluidic device comprising: a base comprising the substrate ofany preceding embodiment; and walls disposed on a top surface of saidbase; wherein the base and the walls together define a microfluidiccircuit.

74. The microfluidic device of any preceding embodiment furthercomprising a cover, wherein the cover is disposed on the walls.

75. The microfluidic device of any preceding embodiment, wherein thewalls comprise a structural polymer.

76. The microfluidic device of any preceding embodiment, wherein thepolymer comprises silicon.

77. The microfluidic device of any preceding embodiment, wherein thepolymer comprises polydimethylsiloxane (PDMS) or photo-patternablesilicone (PPS).

78. The microfluidic device of any preceding embodiment, wherein thecover comprises a structural polymer contained in the walls.

79. The microfluidic device of any preceding embodiment, wherein thewalls comprises SU-8.

80. The microfluidic device of any preceding embodiment, wherein thewalls have a height of at least 30 microns.

81. The microfluidic device of any preceding embodiment, wherein thewalls have a height of about 50-100 microns.

82. The microfluidic device of any preceding embodiment, wherein themicrofluidic circuit comprises one or more microchannels.

83. The microfluidic device of any preceding embodiment, wherein themicrofluidic circuit comprises at least one microchannel and a pluralityof chambers, wherein each chambers opens off of one of themicrochannels.

84. A method of moving a droplet in a microfluidic device of anypreceding embodiment, the method comprising: disposing a droplet of anaqueous solution on a top surface of a base of the microfluidic device;applying a AC voltage potential between the planar electrode and themesh ground electrode; directing structured light at a position on thetop surface of the base, in a location proximal to the droplet; andmoving the structured light relative to the microfluidic device at arate that induces the droplet to move across the top surface of thebase.

85. The method of any preceding embodiment, wherein the droplet have avolume of 200 nL or less.

86. The method of any preceding embodiment, wherein the AC voltagepotential is about 10 ppV to about 80 ppV.

87. The method of any preceding embodiment, wherein the AC voltagepotential is about 30 ppV to about 50 ppV.

88. The method of any preceding embodiment, wherein the AC voltagepotential has a frequency of about 1 kHz to about 1 MHz.

89. The method of any preceding embodiment, wherein the AC voltagepotential has a frequency of about 5 kHz to about 100 kHz.

90. The method of any preceding embodiment, wherein the AC voltagepotential has a frequency of about 5 kHz to about 20 kHz.

91. The method of any preceding embodiment, wherein the droplet has aconductivity of at least 1 mS/m.

92. The method of any preceding embodiment, wherein the structured lightis moved relative to the microfluidic device at a rate of 0.1 cm/sec orgreater.

93. A process for manipulating a droplet in a microfluidic device, theprocess comprising: filling some or all of a microfluidic circuit of amicrofluidic device of any preceding embodiment with a first liquidmedium; applying an AC voltage potential between a planar electrode anda mesh ground electrode of a base of the microfluidic device;introducing a first droplet of liquid into the microfluidic circuit,wherein the first droplet is immiscible in the first liquid medium; and

moving the first droplet to a desired location within the microfluidiccircuit by applying an electrowetting force to the first droplet.

94. The process of any preceding embodiment, wherein the first liquidmedium is an oil.

95. The process of any preceding embodiment, wherein the first liquidmedium is a silicone oil, a fluorinated oil, or a combination thereof.

96. The process of any preceding embodiment, wherein the applied ACvoltage potential is about 10 ppV to about 80 ppV.

97. The process of any preceding embodiment, wherein the applied ACvoltage potential is about 30 ppV to about 50 ppV.

98. The process of any preceding embodiment, wherein the applied ACvoltage potential has a frequency of about 1 to 100 kHz.

99. The process of any preceding embodiment, wherein the applied ACvoltage potential has a frequency of about 5 to 20 kHz.

100. The process of any preceding embodiment, wherein the microfluidicdevice comprises a droplet generator, and wherein the droplet generatorintroduces the first droplet into the microfluidic circuit.

101. The process of any preceding embodiment, wherein the first dropletcomprises an aqueous solution.

102. The process of any preceding embodiment, wherein the first dropletcomprises at least one micro-object.

103. The process of any preceding embodiment, wherein the at least onemicro-object is a biological micro-object.

104. The process of any preceding embodiment, wherein the biologicalmicro-object is a cell.

105. The process of any preceding embodiment, wherein the aqueoussolution is a cell culture medium.

106. The process of any preceding embodiment, wherein the at least onemicro-object is a capture bead having an affinity for a material ofinterest.

107. The process of any preceding embodiment, wherein the first dropletcomprises two to twenty capture beads.

108. The process of any preceding embodiment, wherein the material ofinterest is a biological cell secretion.

109. The process of any preceding embodiment, wherein the material ofinterest is selected from the group consisting of DNA, genomic DNA,mitochondrial DNA, RNA, mRNA, miRNA, or any combination thereof.

110. The process of any preceding embodiment, wherein the first dropletcomprises a reagent.

111. The process of any preceding embodiment, wherein the reagent is acell lysis reagent.

112. The process of any preceding embodiment, wherein the reagentcomprises a non-ionic detergent.

113. The process of any preceding embodiment, wherein the non-ionicdetergent is at a concentration of less than 0.2%.

114. The process of any preceding embodiment, wherein the reagent is aproteolytic enzyme.

115. The process of any preceding embodiment, wherein the proteolyticenzyme is inactivatable.

116. The process of any preceding embodiment, further comprising:introducing a second droplet of liquid into the microfluidic circuit,wherein the liquid of the second droplet is immiscible in the firstliquid medium but miscible with the liquid of the first droplet; movingthe second droplet to a location within the microfluidic circuitadjacent to the first droplet by applying an electrowetting force to thesecond droplet; and merging the second droplet with the first droplet toform a combined droplet.

117. The process of any preceding embodiment, wherein the second dropletis merged with the first droplet by applying an electrowetting force tothe second and/or the first droplet.

118. The process of any preceding embodiment, wherein the first dropletcomprises a biological cell and the second droplet comprises a reagent.

119. The process of any preceding embodiment, wherein the reagentcontained in the second droplet is selected from the group consist of alysis buffer, a fluorescent label, and a luminescent assay reagent.

120. The process of any preceding embodiment, wherein the reagentcontained in the second droplet is a lysis buffer, and wherein saidbiological cell is lysed upon merger of the first droplet and the seconddroplet.

121. The process of any preceding embodiment, wherein applying anelectrowetting force to move and/or merge droplets comprises changing aneffective electrowetting characteristic of a region of the top surfaceof the base of the microfluidic device proximal to the droplet(s).

122. The process of any preceding embodiment, wherein changing aneffective electrowetting characteristic comprises activatingelectrowetting electrodes in a photoconductive layer of the base of themicrofluidic device proximal to the droplet(s).

123. The process of any preceding embodiment, wherein activating theelectrowetting electrodes in the photoconductive layer of the base ofthe microfluidic device proximal to the droplet(s) comprises directing apattern of light into the photoconductive layer of the base of themicrofluidic device proximal to the droplet(s).

Although the description herein contains many details, these should notbe construed as limiting the scope of the disclosure but as merelyproviding illustrations of some of the presently preferred embodiments.Therefore, it will be appreciated that the scope of the disclosure fullyencompasses other embodiments which may become obvious to those skilledin the art.

In the claims, reference to an element in the singular is not intendedto mean “one and only one” unless explicitly so stated, but rather “oneor more.” All structural, chemical, and functional equivalents to theelements of the disclosed embodiments that are known to those ofordinary skill in the art are expressly incorporated herein by referenceand are intended to be encompassed by the present claims. Furthermore,no element, component, or method step in the present disclosure isintended to be dedicated to the public regardless of whether theelement, component, or method step is explicitly recited in the claims.No claim element herein is to be construed as a “means plus function”element unless the element is expressly recited using the phrase “meansfor”. No claim element herein is to be construed as a “step plusfunction” element unless the element is expressly recited using thephrase “step for”.

1. A substrate comprising: a planar electrode; a photoconductive layer;a dielectric layer; a mesh ground electrode; and a hydrophobic coating;wherein the photoconductive layer is interposed between the planarelectrode and the dielectric layer, with a bottom surface of thephotoconductive layer adjoining a top surface of the planar electrodeand a top surface of the photoconductive layer adjoining a bottomsurface of the dielectric layer; wherein the mesh ground electrodeadjoins a top surface of the dielectric layer, wherein the planarelectrode and the mesh ground electrode are configured to be connectedto an AC voltage source; and wherein, when the planar electrode and themesh ground electrode are connected to opposing terminals of the ACvoltage source, the substrate is capable of applying anopto-electrowetting (OEW) force to aqueous droplets in contact with thehydrophobic coating.
 2. The substrate of claim 1, wherein the substrateforms all or part of the base of a microfluidic device.
 3. The substrateof claim 1, wherein the photoconductive layer comprises hydrogenatedamorphous silicon (a-Si:H).
 4. The substrate of claim 1, wherein thedielectric layer comprises a metal oxide.
 5. The substrate of claim 4,wherein the dielectric layer has a thickness of at least 125 nm and/oran impedance of about 10 kOhms to about 50 kOhms.
 6. The substrate ofclaim 4, wherein the dielectric layer was formed by atomic layerdeposition.
 7. The substrate of claim 1, wherein the dielectric layer isa composite dielectric layer having at least a first dielectric layerand a second dielectric layer, with a bottom surface of the firstdielectric layer adjoining the photoconductive layer.
 8. The substrateof claim 7, wherein the mesh ground electrode is interposed between thefirst dielectric layer and the second dielectric layer.
 9. The substrateof claim 7, wherein the first and second dielectric layers both comprisea metal oxide.
 10. The substrate of claim 9, wherein the first andsecond dielectric layers are both formed by atomic layer deposition. 11.The substrate of claim 7, wherein the composite dielectric layer has athickness of about 125 nm to about 175 nm and/or an impedance of about10 kOhms to about 50 kOhms.
 12. The substrate of claim 1, wherein themesh ground electrode comprises wires that are arranged in a latticeshape.
 13. The substrate of claim 12, wherein the mesh ground electrodecomprises gold or aluminum.
 14. The substrate of claim 12, wherein themesh ground electrode comprises aluminum that has an oxidized outersurface.
 15. The substrate of claim 12, wherein the mesh groundelectrode has a linear fill factor β less than or equal to 10%.
 16. Thesubstrate of claim 15, wherein wires of the mesh ground electrode have apitch of about 200 microns to about 500 microns.
 17. The substrate ofclaim 1, wherein the hydrophobic coating has a bottom surface thatadjoins at least a portion of a top surface of the dielectric layer, andoptionally, wherein the bottom surface of the hydrophobic coatingfurther adjoins a top surface of the mesh ground electrode.
 18. Thesubstrate of claim 17, wherein the hydrophobic coating comprises anorganofluorine polymer having at least one perfluorinated segment. 19.The substrate of claim 17, wherein the hydrophobic layer comprises adensely packed monolayer of amphiphilic molecules covalently bonded tomolecules of the dielectric layer.
 20. The substrate of claim 17,wherein the amphiphilic molecules of the hydrophobic layer eachcomprise: a siloxane group, and wherein the siloxane groups arecovalently bonded to the molecules of the dielectric layer; a phosphonicacid group, and wherein the phosphonic acid groups are covalently bondedto the molecules of the dielectric layer; or a thiol group, and whereinthe thiol groups are covalently bonded to the molecules of thedielectric layer and/or the mesh ground electrode.
 21. The substrate ofclaim 20, wherein the amphiphilic molecules of the hydrophobic layercomprise long-chain hydrocarbons having a chain of at least 16 carbons.22. The substrate of claim 20, wherein the amphiphilic molecules of thehydrophobic layer comprise fluorinated carbon chains.
 23. The substrateof claim 22, wherein the fluorinated carbon chains have the chemicalformula CF₃—(CF₂)_(m)—(CH₂)_(n)—, wherein m is at least 2, n is at least2, and m+n is at least
 9. 24. The substrate of claim 20, wherein thehydrophobic layer is patterned such that select regions are relativelyhydrophilic compared to the remainder of the hydrophobic layer.
 25. Amicrofluidic device comprising: a base comprising the substrate of claim1; and walls disposed on a top surface of said base; wherein the baseand the walls together define a microfluidic circuit.
 26. Themicrofluidic device of claim 25 further comprising a cover, wherein thecover is disposed on the walls.
 27. The microfluidic device of claim 25,wherein the microfluidic circuit comprises at least one microchannel anda plurality of chambers, wherein each chambers opens off of one of themicrochannels.
 28. A method of moving a droplet in a microfluidic deviceof claim 25, the method comprising: disposing a droplet of an aqueoussolution on a top surface of a base of the microfluidic device; applyinga AC voltage potential between the planar electrode and the mesh groundelectrode; directing structured light at a position on the top surfaceof the base, in a location proximal to the droplet; and moving thestructured light relative to the microfluidic device at a rate thatinduces the droplet to move across the top surface of the base.
 29. Themethod of claim 28, wherein the AC voltage potential is about 10 ppV toabout 80 ppV, or about 30 ppV to about 50 ppV.
 30. The method of claim29, wherein the AC voltage potential has a frequency of about 1 kHz toabout 1 MHz, about 5 kHz to about 100 kHz, or about 5 kHz to about 20kHz.